BAFF selective binding compounds and related methods

ABSTRACT

The present invention relates to peptide antagonists that bind with high specificity and affinity to B-Lymphocyte stimulator (“BAFF”), thereby antagonizing BAFF receptor (“BAFF-R”) signaling. The invention more specifically relates to VNAR single chain antibodies derived from nurse shark that bind to BAFF, BAFF antagonist compounds and compositions comprising a BAFF specific VNAR binding moiety, methods for preparing them, diagnostic and therapeutic methods of use relating to in vitro or in vivo B cell depletion, e.g., to treat and/or prevent a pathological condition, disorder or disease in which it is beneficial to kill or deplete B cells, such as in autoimmune diseases including systemic lupus erythematosus (SLE), rheumatoid arthritis (RA) or multiple sclerosis (MS), and in certain hematological cancers, including lymphomas, leukemias and myelomas.

CROSS REFERENCE TO RELATED APPLICATION

This application is a divisional application of U.S. Ser. No.15/107,921, filed on Jun. 24, 2016, which is a national stage filingunder 35 U.S. § 371 of Intl. Appln. No. PCT/US2014/071913, filed Dec.22, 2014, which claims the benefit of U.S. Application Ser. No.61/920,591, filed on Dec. 24, 2013, each of which is incorporated hereinby reference in its entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Dec. 18, 2014, isnamed OSX1301-WO1_SL.txt and is 52,715 bytes in size.

FIELD OF THE INVENTION

The present invention relates to peptide antagonists that bind with highspecificity and affinity to B-Lymphocyte stimulator (“BAFF”), therebyantagonizing BAFF receptor (“BAFF-R”) signaling. The invention morespecifically relates to VNAR single chain antibodies derived from nurseshark that bind to BAFF, BAFF antagonist compounds and compositionscomprising a BAFF specific VNAR binding moiety, methods for preparingthem, diagnostic and therapeutic methods of use relating to in vitro orin vivo B cell depletion, e.g., to treat and/or prevent a pathologicalcondition, disorder or disease in which it is beneficial to kill ordeplete B cells, such as in autoimmune diseases including systemic lupuserythematosus (SLE), rheumatoid arthritis (RA) or multiple sclerosis(MS), and in certain hematological cancers, including lymphomas,leukemias and myelomas.

BACKGROUND OF THE INVENTION

BAFF-R (also called BR3, TNFRSF13C or CD268) is a tumor necrosis factor(TNF) receptor superfamily member expressed predominantly on matureB-lymphocytes and on a subset of T-cells (L. G. Ng, et al., J Immunol,173 (2004), pp. 807-817. BAFF-R and two other TNF superfamily receptorscalled TACI (transmembrane activator and calcium modulator andcyclophilin ligand interactor) and BCMA (B-cell maturation antigen), areexpressed mainly on B lymphocytes and their expression varies as afunction of B cell maturation. Each specifically binds a ligand calledB-Lymphocyte stimulator (BAFF; also referred to as BLyS, CD257, TALL-1,THANK, TNFSF13B or ZTNF4) which is expressed in myeloid cells and avariety of other cell types. While BCMA and TACI also interact withother ligands, BAFF-R is reportedly exclusive to BAFF. Each of the threeTNF receptors has a different binding affinity for BAFF. BAFF trimerizesand binds to cell surface BAFF-R, upon which the complex is internalizedby receptor-mediated endocytosis.

The BAFF/BAFF-R interaction has been shown to be significant in B-cellsurvival, maintenance and proliferation. Functionally, the BAFF/BAFF-Rinteraction is critical for maturation of immature transitional B-cellsand for survival, migration and activation of mature B-cells includingisotype class switching. BAFF can act alone or in concert with otheragents, e.g., B-cell receptor (BCR), interleukin-4, interleukin-21 orCD40 ligand.

BAFF antagonists may have therapeutic benefit for treating autoimmunediseases in which B cells play a pathogenic role. Overproduction of BAFFcan trigger severe autoimmune disorders in mice resembling systemiclupus erythematosus (SLE) and Sjogren's syndrome (SS) (Ju et al.,Immunol. 2007120(2):281-9). Increased levels of BAFF are also found inhuman patients suffering from SLE, SS, rheumatoid arthritis (RA),Wegener's granulomatosis and certain B-cell malignancies. Moreover, inanimal models of autoimmune disease, such as SLE, rheumatoid arthritis(e.g., collagen-induced arthritis) and multiple sclerosis (e.g.,experimental autoimmune encephalomyelitis), the disease phenotype can bepartially reverted by treating with soluble BAFF-R-Fc fusion proteinsthat bind to BAFF thereby antagonizing BAFF/BAFF-R interaction.Treatment with BAFF-R-Fc fusion proteins has also been shown to inhibitchronic graft-versus-host disease (cGVHD) by blocking B-cell survival.And, anti-BAFF antibodies are clinically beneficial when administered torheumatoid arthritis or SLE patients, establishing a nexus between BAFFantagonism and therapeutic efficacy in these autoimmune disorders.

A variety of B-cell malignancies show increased expression of BAFF-R.Different Non-Hodgkin's Lymphoma (NHL) cell lines, for example, expressBAFF-R to different degrees. The BAFF/BAFF-R interaction also increasesthe survival and proliferation of malignant cells, enabling cancer cellsto proliferate faster than normal B-cells. Because BAFF-R is thought tobe the only receptor that mediates the B cell survival signal from BAFF,agents that modulate BAFF/BAFF-R interaction could be useful treatmentsfor various B cell malignancies.

Accordingly, BAFF-R, as the predominant BAFF receptor expressed on Bcell lines, is thought to represent an attractive target for therapeuticintervention in B cell malignancies such as lymphomas and in autoimmunediseases involving B cells. To that end, a number of B-cell targetingtherapeutic antibodies have been developed, the earliest ones directedto CD20 (e.g., rituximab, a chimeric mouse/human IgG1 approved forhematological cancers and refractory RA; ofatumumab, a human IgG1approved for refractory chronic lymphocytic leukemia “CLL” and inclinical trials for other hematological cancers, including relapsingremitting multiple sclerosis “RRMS”; and ocrelizumab, a humanized IgG1in clinical trials for RRMS). Epratuzumab is a humanized mousemonoclonal antibody directed to CD22, currently in clinical trials forsystemic lupus erythematosus “SLE” and Non-Hodgkin's Lymphoma “NHL”.Certain human anti-BAFF antibodies have been developed, includingbelimumab (human IgG1) and blisibimod (human IgG4), which are approvedand/or in clinical trials for treatment of SLE and various otherindications. See, e.g., WO2011/160086; WO2006/025345; WO2003/016468; andWO2000/043032.

Monoclonal antibodies have revolutionized biotechnology and are now keytherapeutic drugs in the treatment of human disease. Despite theirsuccesses, therapeutic monoclonal antibodies have certain limitations,such as restricted activity against certain types of antigen, poortissue penetration, unwanted effector function in many situations, thecost of manufacturing, product instability and aggregation. Singledomain antibodies that occur naturally in the shark are particularlyattractive for the development of next generation biotherapeutics. VNARsare small (12 kDa), stable, soluble, monomeric antigen-binding domainsthat can be configured into many different therapeutic modalities. Theisolation of various VNAR based binding moieties has been described.See, e.g., WO2003/014161 and WO2005/118629.

It would be desirable to have additional BAFF antagonists, especiallyones having one or more advantageous biological properties withtherapeutic and/or diagnostic benefit over current anti-BAFF antibodies.

SUMMARY OF THE INVENTION

Complex phage libraries have been generated using a shark VNAR derivedscaffold which enables the generation of novel therapeutic products, inparticular, specific binding moieties which bind selectively and withhigh affinity to human BAFF antigen, thereby producing a BAFF antagonistcompound. The invention thus provides BAFF specific binding moieties andBAFF antagonist compounds comprising them. BAFF specific bindingmoieties comprise a CDR1 region and a CDR3 region interspersed by aframework region FW2-3. CDR1 and CDR3 regions are also bordered byframework regions FW1 and FW4, respectively. The CDR1 region comprisesor consists essentially of a peptide having an amino acid sequence offormula D-X₂-X₃-X₄-A-L-X₇ (SEQ ID NO: 1) in which X₂ is N or S; X₃ is N,I or S; X₄ is C or Y; and X₇ is S, P or G. The CDR3 region comprises orconsists essentially of a peptide having an amino acid sequence offormula (a) D-X_(a)-L-Z₍₁₋₆₎-C(SEQ ID NO: 2) or formula (b)C-Z₍₁₋₆₎-D-X_(a)-L (SEQ ID NO: 3) in which X_(a) is selected from W, P,R, V or L; and Z₍₁₋₆₎ is a stretch of any one to six amino acidresidues. Specific framework and CDR region amino acid sequences areprovided, and compounds comprising combinations of each region areincluded. BAFF antagonist compounds compete with or inhibit one or morebioactivities of a native BAFF ligand in vitro or in vivo. Nucleic acidsequences encoding one or more BAFF specific binding moieties, vectorscomprising nucleic acid sequences, and host cells comprising them arealso provided, as are related methods for producing a BAFF antagonistcompound.

BAFF specific binding moieties or BAFF antagonist compounds may be usedto produce variants and derivatives, including conjugates, e.g.,immunoconjugates. The antagonist compounds, and variants or derivativesthereof, may be combined with other therapeutic agents in compositionsfor use in related therapeutic, prophylactic and diagnostic methods.Therapeutic methods are provided for treating a B cell relatedcondition, disease or disorder associated with a pathology in B cellproliferation, maturation or maintenance; immunoglobulin production,such as a B cell malignancy or a B-cell related autoimmune condition.Methods for identifying, quantifying or localizing a BAFF-R containingbiological sample are also provided, as are methods for the targeteddelivery of a payload to a BAFF-R expressing cell using a BAFF specificbinding moiety-payload conjugate.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the protein sequence of the ten templates used in the sharklibrary design. The mutations are highlighted. FIG. 1 discloses SEQ IDNOS 43-53, respectively, in order of appearance.

FIG. 2 is a schematic of the overlap PCR principle, in which sequencevariability is introduced in both the VNAR frameworks and the CDR3.

FIG. 3 shows the generic oligonucleotide design used in the librarybuilding. A set of six oligonucleotides (SEQ ID NOS 55, 56, 55, 56, 55and 56 respectively, in order of appearance) was used for each CDR3length in order to cover all sequence permutations on the edges of theCDR3. FIG. 3 discloses the left peptide as SEQ ID NO: 54 and the rightside peptides as SEQ ID NOS 57, 57, 58, 58, 59 and 59, respectively, inorder of appearance.

FIG. 4 shows selected templates and the randomization approach.Highlighted residues were conserved. FIG. 4 discloses SEQ ID NOS 60-65,respectively, in order of appearance.

FIG. 5 shows the percentage of eluted phages after each round ofselection (see Example 2). The number of phages eluted from theBAFF-coated wells after each round of selection is indicated as apercentage of the amount of phages that were incubated on the plate.

FIG. 6 shows polyclonal phage ELISA on selection outputs from thelibrary constructed according to Example 1. Phages (1×10¹²) from eachround's input were incubated in microwells coated with either human BAFF(hBAFF), or negative VNAR controls (hTfR or HSA). After washing steps,bound phages were detected and quantified using a specific anti-M13antibody.

FIG. 7 shows results from screening for hBAFF binding clones. Eighteenunique clones (based on their DNA sequences) were selected for bindingspecifically to hBAFF in an ELISA format (see Example 2; threshold=fourtimes negative VNAR control, HSA).

FIG. 8 shows results from screening for hBAFF blocking clones. Fourteenof the eighteen binding clones are capable of inhibiting BAFF/BAFF-R(BLys) binding by at least 50% in a blocking ELISA format. Negativecontrols are shown in white. Clones that inhibit BAFF/BAFF-R binding byless than 50% are shown in grey (see Example 2).

FIG. 9 shows EC50 curves of five selected clones. HSA-1B8 is anon-specific VNAR used as a negative control (see Example 3).

FIG. 10 shows IC50 curves of five selected clones. HSA-1B8 is anon-specific VNAR used as a negative control (see Example 3).

FIG. 11 shows inhibition of BAFF bioactivity in mouse splenocytes. Theresults depict the cellular IC50 curves of the five best inhibitors ofBAFF bioactivity. HSA-1B8 is a non-specific VNAR used a negative control(see Example 5).

FIG. 12 shows the ability of periplasmic protein extracts from thirteendifferent VNAR clones to block BAFF binding to the TACI (dark gray) andBCMA (light gray) receptors as tested by ELISA (see Example 6). Alltested VNAR clones block the interaction between BAFF and both receptorscompared to a negative VNAR control (a-Lys) or to a test sample with noadded VNAR (blank).

FIG. 13 shows the ability of periplasmic protein extracts from thirteendifferent VNAR clones to bind directly to either BAFF-Fc (black bars), arelated TNF family ligand, APRIL (gray bars) or human serum albumin(HSA; white bars), as tested by ELISA (see Example 7). With theexception of B07, which showed weak cross-reactivity, all other VNARclones appear to bind specifically to BAFF.

FIG. 14 shows the ability of periplasmic protein extracts from thirteendifferent VNAR clones to compete for binding with VNARs formatted as Fcfusions, as tested by ELISA (see Example 8). Each VNAR clone was testedagainst two VNAR clones formatted as Fc fusions: Fc-A07 (dark gray bars)and Fc-B07 (black bars). Fc-5A7 (an unrelated Fc fusion; light graybars) does not compete for BAFF binding and was comparable to the mockcontrol (white bars). Every BAFF specific VNAR clone was competed byboth A07 and B07 Fc fusions but not by the negative control (Fc-5A7),indicating that all of the VNARs target a similar or overlapping epitopeon BAFF.

FIGS. 15A-B show biochemical IC50 curves of five different VNAR monomers(ID2-101A05, -101A09, -101B07, -101A07, and -101B10) serially diluted inthe presence of constant (0.5 nM) BAFF-Fc and their ability to bind to(A) TACI and (B) BCMA receptors compared to BAFFr (BR3) (see Example 9).

FIGS. 16A-B show the species cross-reactivity of two VNAR clones (A07,circles; and B07, squares) formatted as a human Fc fusion moleculescompared to the positive (BAFFr; downward pointing triangles) andnegative (5A7-Fc; upward pointing triangles) controls. A07 and B07exhibited similar potencies against human BAFF but A07 was more potentin blocking mouse BAFF activity in vitro (see Example 10).

FIG. 17 shows characterization by flow cytometry of the expression ofB-cell subset markers on splenocytes isolated from mice injected (i.p.)with 100 μg of A07-Fc at days 0 and 5, and isolated on day 8. Micetreated with A07-Fc (A07) showed a pronounced reduction in the number oflate transitional B cells (T3) compared to untreated control mice(Ctrl.) (see Example 11).

FIG. 18 shows binding potency of anti-BAFF VNAR A07 formatted on a mouseFc having effector function. The EC50 of A07-mFc was estimated at 0.6 nMbased on the ELISA binding curve, whereas the 5A7-mFC control failed tobind BAFF (see Example 12).

DETAILED DESCRIPTION OF THE INVENTION

In order that the present invention may be more readily understood,certain terms are defined below. Additional definitions may be foundwithin the detailed description of the invention.

Throughout this specification, the word “comprise” or variations such as“comprises” or “comprising” will be understood to imply the inclusion ofa stated integer (or components) or group of integers (or components),but not the exclusion of any other integer (or components) or group ofintegers (or components).

The singular forms “a,” “an,” and “the” include the plurals unless thecontext clearly dictates otherwise.

The term “including” is used to mean “including but not limited to.”“Including” and “including but not limited to” are used interchangeably.

The terms “patient,” “subject,” and “individual” may be usedinterchangeably and refer to either a human or a non-human animal. Theseterms include mammals such as humans, primates, livestock animals (e.g.,bovines, porcines), companion animals (e.g., canines, felines) androdents (e.g., mice and rats).

As used herein, “treating” or “treatment” and grammatical variantsthereof refer to an approach for obtaining beneficial or desiredclinical results. The term may refer to slowing the onset or rate ofdevelopment of a condition, disorder or disease, reducing or alleviatingsymptoms associated with it, generating a complete or partial regressionof the condition, or some combination of any of the above. For thepurposes of this invention, beneficial or desired clinical resultsinclude, but are not limited to, reduction or alleviation of symptoms,diminishment of extent of disease, stabilization (i.e., not worsening)of state of disease, delay or slowing of disease progression,amelioration or palliation of the disease state, and remission (whetherpartial or total), whether detectable or undetectable. “Treatment” canalso mean prolonging survival relative to expected survival time if notreceiving treatment. A subject (e.g., a human) in need of treatment maythus be a subject already afflicted with the disease or disorder inquestion. The term “treatment” includes inhibition or reduction of anincrease in severity of a pathological state or symptoms relative to theabsence of treatment, and is not necessarily meant to imply completecessation of the relevant disease, disorder or condition.

As used herein, the terms “preventing” and grammatical variants thereofrefer to an approach for preventing the development of, or altering thepathology of, a condition, disease or disorder. Accordingly,“prevention” may refer to prophylactic or preventive measures. For thepurposes of this invention, beneficial or desired clinical resultsinclude, but are not limited to, prevention or slowing of symptoms,progression or development of a disease, whether detectable orundetectable. A subject (e.g., a human) in need of prevention may thusbe a subject not yet afflicted with the disease or disorder in question.The term “prevention” includes slowing the onset of disease relative tothe absence of treatment, and is not necessarily meant to implypermanent prevention of the relevant disease, disorder or condition.Thus “preventing” or “prevention” of a condition may in certain contextsrefer to reducing the risk of developing the condition, or preventing ordelaying the development of symptoms associated with the condition.

As used herein, an “effective amount,” “therapeutically effectiveamount” or “effective dose” is an amount of a composition (e.g., atherapeutic composition or agent) that produces at least one desiredtherapeutic effect in a subject, such as preventing or treating a targetcondition or beneficially alleviating a symptom associated with thecondition.

As used herein, the term “BAFF-R” or “BAFF receptor” refers to amammalian BAFF-R, unless the context indicates that it refersspecifically to human BAFF-R, as disclosed in, e.g., WO2000/04032,WO2001/012812, WO2001/060397 and WO2002/024909. WO2006/073941 andWO2010/007082 refer to anti-BAFF-R antibodies in general. WO2006/073941describes specific anti-BAFF-R antibodies.

VNAR Semi-Synthetic Library Construction and Screening

A Type 2 nurse shark VNAR semi-synthetic library was constructed by arationale design based on sequence analysis of 189 Type 2 VNAR sequencescontaining a single cysteine in their CDR3 region (see M. Diaz, et al.,Immunogenetics 54 (2002) pp. 501-512). These sequences were obtained byrandomly sequencing clones in naïve VNAR libraries built from twodifferent adult nurse sharks. Information obtained by alignment of the189 protein sequences was used to design a new semi-synthetic libraryincluding sequence variation in both the CDR3 and the framework regions.

The collected information included:

(i) The length of the CDR3s: It was observed that more than 80% of thenaturally occurring type 2 CDR3s have a length ranging from 11 to 18amino acids. These 8 different lengths were therefore chosen to buildthe semi-synthetic library.

(ii) The position of the single cysteine in the CDR3: The amino acidcomposition at each position of the CDR3 was analysed and the preferredposition of the single cysteine determined in the 8 selected CDR3lengths. This information was incorporated in the library design byeither fixing a single cysteine residue in the CDR3 (using a TGC codon),or by using a “loose” cysteine approach by which the preferred cysteineposition, as well as the two immediately adjacent residues, were mutatedto a DRY degenerate codon (1/6 chance to form a cysteine).(iii) The presence of fixed residues in CDR3: The same sequence analysisrevealed that the amino acids DV were predominantly found at the lasttwo positions of the CDR3. This sequence information was incorporatedinto the library design by fixing these two amino acids residues.(iv) The most commonly found mutations in the framework regions: Theanalysis of the amino acid composition of the 189 VNAR backbones allowedidentification of the most frequently found amino acid substitutions atevery position of the framework regions. The most frequent mutationswere then introduced in the library design using a mixture of tenselected framework templates accumulating a number of frequently foundmutations (FIG. 1) in the PCR reaction.(v) The sequence conservation on the edges of the CDR3: The sameframework analysis allowed identification of specific sequencevariations on the edges of the CDR3 region. It was observed that shorterCDR3s (less than 16 amino acids) were usually preceded by the CNVsequence, while longer CDR3s (more than 16 amino acids) were usuallypreceded by the CKV sequence. This sequence variation was thereforeincorporated in the library design by fixing amino acids at thesepositions. Three main sequence permutations were also observed inC-terminus of the CDR3. These sequence variations were also included inthe library design by synthesising three sets of oligonucleotidesincorporating each amino acid permutation.

The VNAR library was generated by overlap PCR (FIG. 2) incorporating allof the above information. A mixture of the ten selected templates wasused to introduce framework mutations, while a mixture ofoligonucleotides was used to incorporate both randomization of the CDR3by NNK codons, fixed and loose cysteine residues by use of TGC and DRYcodons, and sequence variability on both edges of the CDR (FIGS. 3-4).

The main differences with previously built libraries are that: (i) anurse shark backbone was used; (ii) selected mutations were introducedin the framework regions; and (iii) amino acids permutations, reflectingnaturally occurring variations, were introduced on the edges of theCDR3s.

A Type 1 nurse shark VNAR semi-synthetic library was built from threespecific clones identified by randomly sequencing VNARs in naïvelibraries built from two different adult nurse sharks. These clonesharboured unusually long CDR3 regions of 26 and 32 amino acids and hadvery few framework mutations (FIG. 4). In order to generate asemi-synthetic library specifically enriched for clones harbouring longCDR3s, the CDR3 of these 3 clones was randomized, as previouslydescribed, by overlap PCR, keeping only the two cysteine residuesunchanged in order to preserve the structural integrity of the molecule.

The main differences with previously built libraries are that: (i) anurse shark backbone was used; (ii) a VNAR Type 1 library was used; and(iii) the library is biased for extended CDR3s.

Isolating BAFF Binding VNAR Sequences

VNARs capable of specifically and selectively binding to human BAFF(hBAFF) were isolated by four rounds of selection and amplification of asemi-synthetic phage display library on immobilized hBAFF recombinantprotein (Example 2). In order to select for high affinity clones, thestringency of selection was increased at each round by decreasing hBAFFconcentration and increasing the number of washing steps. The efficiencyof the selection procedure was assessed by plotting the percentage ofeluted phages after each selection round. A 3000-fold increase in phagerecovery was observed between rounds 2 and 4 (FIG. 5).

The BAFF binding specificity of selected phages was confirmed by apolyclonal phage ELISA in which three different coatings were used(BAFF, hTfR, and HSA). A strong increase in binding phage was observedfrom round 3, specifically on the BAFF-coated surface and not on othercoatings (FIG. 6).

Screening for hBAFF Binding Clones

In order to test the ability of individual clones to bind hBAFF, 93independent clones were randomly selected in the outputs of both roundsthree and four (Example 2). The periplasmic fraction of these clones(containing monomeric VNAR molecules) was exposed to a hBAFF-coatedsurface (in an ELISA format) and the bound VNARs were detected using aspecific antibody. More than 90% of the selected clones appearedhBAFF-specific using this method, with no notable background on aHSA-coated surface.

The DNA sequence of positive clones was determined and the bindingclones were sorted into categories based on their sequence identities.Eighteen unique sequences were selected this way, using a threshold ofhigher than four for the ratio of BAFF-binding signal to HSA-bindingsignal (FIG. 7). Interestingly, the vast majority of binding clonesappear to harbour a DXL motif in their CDR3, just upstream of the singlecysteine (see Table I, below). This motif was previously described asbeing part of the main binding site of BAFF to its receptor.

TABLE 1 Protein sequence of isolated BAFF binding VNARs(DXL motifs are highlighted in bold, CDR3 cysteine(s) are underlined)Clone SEQ ID2- ID NO: FW1 CDR1 FW2-3 CDR3 FW4 101A05  4 AQAAARVDQTPQTITDNNCALS TTYWYRKKSGSTNEENISKGGRYVETV SKDWLLCRDR YGDGTAVTVNAASGAHHKETGESLTINCVLR NSGSKSFSLRINDLTVEDSGTYRCKV GRRETDV HHHHGADYKDDDDK* 101A07 5 AQAAARVDQTPQTIT DSNCALS NLYWYRKKSGSTNEESISLGGRYVETV QLPYDPLTKEYGDGTAVTVNAASGAHH KETGESLTINCVLR NSGSKSFSLRINDLTVEDSGTYRCKV CILGRMDVHHHHGADYKDDDDK* 101A01  6 AQAAARVDQTPQTIT DSNCALSNLYWYRKKSGSTNEESISLGGRYVETV RRARVIGGEY YGGGTAVTVNAASGAHH KETGESLTINCVLRNSGSKSFSLRINDLTVEDSGTYRCKV CRVQWQDV HHHHGADYKDDDDK* 101A03  7AQAAARVDQTPQTIT DNNCALS TTYWYRKKSGSTNEENISKGGRYVETV RVDRLLCGWRYGGGIVVTVNAASGAHH KETGESLTINCVLR NSGSKSFSLKINDLTVEDSGTYRCKV VGRRQLGDVHHHHGADYKDDDDK* 101A09  8 AQAAARVDQTPQTIT DNNCALSTTYWYRKKSGSTNEESISKGGRYVETV REDPLMCRYY YGGGTVVTVNAASGAHH KETGESLTINCVLRNSGSKSFSLRINDLTVEDSGTYRCKV LDRYRDV HHHHGADYKDDDDK* 101B07  9AQAAARVDQTPRSVT DSICALS STHWYRKKSGSTNEESISKGGRYVETV HGGRSTGLCGYGGGTAVTVNAASGAHH KETGESLTINCVLR NSGSKSFSLRINDLTVEDSGTYRCKV DVLLAGDVHHHHGADYKDDDDK* 101B08 10 AQAAARVDQTPQTIT DSNCALSSTLWYRTKSGSRNEESISKGGRYVETV PRDLLLCKRP YGGGTAVTVNAASGAHH KETGESLTINCVLRNSGSKSFSLKINDLTVEDSGTYRCKV RARLPDV HHHHGADYKDDDDK* 101A12 11AQAAARVDQTPQTVT DASYALG STCWYRKKSGSRNEESISKGGRYVETV RDPLLFPRDRYGGGTVVTVNAASGAHH KETGESLTINCVLR NSGSKSFSLRINDLTVEDSGTYRCKV CDGESKDVHHHHGADYKDDDDK* 101B09 12 AQAAARVDQTPQTIT DSNCALPSTYWYRKKSGSTNEESISLGGRYVETV LSNVHICCRF YGDGTAVTVNAASGAHH KETGESLTINCVLRNSGSKSFSLRINDLTVEDSGTYRCKV GSCADV HHHHGADYKDDDDX* 101B11 13AQAAARVDQTPRSVT DSNCALS STYWYRKKSGSTNEENISKGGRYVETV MLDPLLCPALYGGGTAVTVNAASGAHH KETGESLTINCVLR NSGSKSFSLRINDLTVEDSGTYRCKV LESMTDVHHHHGADYKDDDDK* 101B01 14 AQAAARVDQTPQTIT DSNCALSSTYWYRKKSGSTNEESISKGGRYVETV APTIISGCSI YGGGTAVTVNAASGAHH KETGESLTINCVLRNSGSKSFSLRINDLTVEDSGTYRCNV KRRDV HHHHGADYKDDDDK* 101B10 15AQAAARVDQTPQTIT DSNCALS STYWYRKKSGSTNEESISKGGRYVETV RIDPLLCNASYGGGTVVTVNAASGAHH KETGESLTINCVLR NSGSKSFSLRINDLTVEDSGTYRCKV YVKWDDVHHHHGADYKDDDDK* 101B05 16 AQAAARVDQTPRSVT DSICALSSTHWYRKKSGSTNEESISKGGRYVETV NHDLLTSSRR YGGGTVVTVNAASGAHH KETGESLTINCVLRNSGSKSFSLRINDLTVEDSGTYRCKV CQSQIKDV HHHHGADYKDDDDK* 101B03 17AQAAARVDQTPRSVT DNNCALS TTYWYRKKSGSTNEENISKGGRYVETV KPDLLFCSSSYGGGTAVTVNAASGAHH KETGESLTINCVLR NSGSKSFSLRINDLTVEDSGTYRCKV GLGLIQDVHHHHGADYKDDDDK* 101B12 18 AQAAARVDQTPQTIT DSNCALSSTYWYRKKSGSTNEESISKGGRYVETV FIDPLLCSRD YGDGTAVTVNAASGAHH KETGESLTINCVLRNSGSKSFSLRINDLTVEDSGTYRCKV ALGFSDV HHHHGADYKDDDDK* 101C02 19AQAAARVDQTPQTIT DNNCALS TTYWYRKKSGSTNEENISKGGRYVETV TRDPLFCSYRYGGGTVVTVNAASGAHH KETGESLTINCVLR NSGSKSFSLKINDLTVKDSGTYRCKV ASKRHDVHHHHGADYKDDDDK* 101C01 20 AQAAARVDQTPQTIT DNNCALSTTYWYRKKSGSTNEENISKGGRYVETV RLDLLLCRNG YGGGTAVTVNAASGAHH KETGESLTINCVLRNSGSKSFSLRINDLTVEDSGTYRCKV STNSIDV HHHHGADYKDDDDK* 101C03 21AQAAARVDQTPRSVT DSICALS STHWYRKKSGSTNEESISLGGRYVETV TRYVVFSGSTYGGGTVVTMNAASGAH KETGESLTINCVLR NSGSKSFSLKINDLTVEDSGTYRCKV CRMRRADVHHHHHGADYKDDDDK*Screening for hBAFF/BAFF-R Blocking Clones

In order to test the ability of the eighteen selected BAFF bindingclones to block the interaction between BAFF and its receptor, BAFF-R, aperiplasmic fraction from each clone was pre-incubated with recombinanthBAFF before being exposed to a surface coated with BAFF-R. Ability toblock the BAFF/BAFF-R interaction was then measured by specificallydetecting the amount of BAFF bound to the plate by using a specificantibody, as described in Example 2. As used herein, a blocking clone isone that is capable of inhibiting binding of BAFF to its receptor by atleast 50%. Using these criteria, fourteen of the eighteen binding clonesappeared to prevent the BAFF/BAFF-R interaction (FIG. 8; Example 2).

EC₅₀ values may be used as a numerical measure of potency, such as forability to bind with a given binding partner, e.g., a ligand orreceptor. An EC₅₀ value is a measure of the concentration of a compoundrequired to achieve half of that compound's maximal activity in aparticular assay (Example 3). An IC₅₀ value or inhibition constant isthe concentration which inhibits binding of one agent to another agentby 50% and may also be used as a numerical measure of the ability of aBAFF binding moiety or antagonist compound to compete with a differentBAFF binding agent, e.g., an anti-BAFF antibody, for binding to BAFF,e.g., to human BAFF (Example 3).

Binding affinities may be measured as a constant of binding affinity(K_(A)), or as a constant of dissociation from a bound complex (K_(D)).In some embodiments, compounds of the present invention, the K_(A) orK_(D) towards hBAFF is below 20 nM. In some embodiments of compounds ofthe present invention, the K_(A) or K_(D) towards hBAFF is below 10 nM.In further embodiments of compounds of the present invention, the K_(A)or K_(D) towards hBAFF is below 5 nM. In still further embodiments ofcompounds of the present invention, the K_(A) or K_(D) towards hBAFF isbelow 1 nM.

Biological Activities of Selected VNARs

In order to test the ability of selected VNARs to inhibit BAFFbiological activity, each of the lead molecules was tested in a mousesplenocyte survival assay where mouse splenic B cells were exposed toBAFF in the presence or in the absence of a putative BAFF antagonistVNAR. Six of the lead VNARs did not appear to affect BAFF bioactivity.An IC50 was determined for five of the remaining seven lead VNARs,ranging from 60 to 200 nM, which is in the same order of magnitude ashBAFF-R, known to display an IC50 of 120 nM.

A summary of biochemical data of selected BAFF selective VNAR clones isshown in Table 2. These data include EC50 and IC50 measurements as wellas an indication in certain cases as to whether a BAFF antagonistcompound, selected for its ability to bind to hBAFF, exhibits crossreactivity in the mouse, i.e., binds to both human and mouse BAFF. Suchcross-reactive BAFF binding moieties and compounds will be useful inconducting experiments in animals, e.g., mouse B cell related diseasemodels. The following are representative for binding or inhibition ofbinding by a BAFF specific VNAR monomer according to the invention.Therapeutic versions, including variants and derivatives thereof, areexpected to be more potent.

TABLE 2 Biochemical data summary of selected clones ELISA ELISAInhibition Mouse CLONE EC50 IC50 of BAFF CELLULAR cross- NAME (nM) (nM)Bioactivity IC50 (nM) reactivity ID2-101A07 0.5 6.8 ++ 58 ++++ID2-101B07 0.8 6.8 ++ 60 −−−− ID2-101A05 1.5 13.8 ++ 135 ++ ID2-101B10 12.8 ++ ≈100 + ID2-101A09 4.9 9.7 ++ 215 ++ ID2-101C01 2.13 3.85 ++ n.d.n.d. ID2-101A12 4.57 49.16 ++ n.d. n.d. ID2-101B11 2.49 6.85 − n.d. n.d.ID2-101B12 2.7 9.32 − n.d. n.d. ID1-101B09 4.07 36.77 − n.d. n.d.ID2-101B01 7.4 91.2 − n.d. n.d. ID2-101A03 17.16 12.33 − n.d. n.d.ID2-101B05 11.82 148.2 − n.d. n.d.Polypeptide Sequences and Compounds Comprising a BAFF Specific VNAR

The present invention provides a BAFF specific binding moiety, e.g.,polypeptide, and BAFF antagonist compounds comprising BAFF specificbinding moieties. Isolated BAFF binding VNARs are also provided. Incertain embodiments, the BAFF specific binding moiety is specific for amammalian BAFF. In certain embodiments, the BAFF binding moiety isspecific for human BAFF. In certain embodiments, the BAFF specificbinding moiety comprises a DXL motif in its CDR3 region. In certainembodiments, the BAFF specific binding moiety blocks the interactionbetween hBAFF and its receptor, BAFF-R.

In certain embodiments, the BAFF specific binding moiety comprises a CDR1 region and a CDR3 region interspersed by a framework region (see FW2-3in Table 1 and below), in which the CDR 1 region comprises or consistsessentially of a peptide having an amino acid sequence of formula:D-X₂-X₃-X₄-A-L-X₇ (SEQ ID NO: 1)

wherein X₂ is N or S;

X₃ is N, I or S;

X₄ is C or Y; and

X₇ is S, P or G.

In certain aspects of this embodiment, the CDR 1 region, which innaturally-occurring VNARs is a conserved seven amino acid residuestretch, comprises or consists essentially of a peptide selected fromDNNCALS (SEQ ID NO: 22), DSNCALS (SEQ ID NO: 23), DSNCALP (SEQ ID NO:24), DSICALS (SEQ ID NO: 25) or DASYALG (SEQ ID NO: 26) (Table 1).

The CDR3 region in naturally-occurring VNARs is of heterogeneous size,ranging from about 7 to about 32 amino acid residues in length. Insynthetic VNAR libraries exemplifying the present invention, CDR3regions of 11 to 18 residues, and 26 or 32 residues, were constructed.

In certain other embodiments of the invention, the BAFF specific bindingmoiety comprises a CDR 1 region and a CDR3 region interspersed by aframework region (see FW2-3 in Table 1 and below), wherein the CDR 3region comprises a peptide having an amino acid sequence of formula (a)D-X_(a)-L-Z₍₁₋₆₎-C(SEQ ID NO: 2);

wherein X_(a) is selected from W, P, R, V or L; and Z₍₁₋₆₎ is a stretchof any one to six amino acid residues.

In certain other embodiments of the invention, the BAFF specific bindingmoiety comprises a CDR 1 region and a CDR3 region interspersed by aframework region (see FW2-3 in Table 1 and below), wherein the CDR 3region comprises a peptide having an amino acid sequence of formula (b)C-Z₍₁₋₆₎-D-X_(a)-L (SEQ ID NO: 3);

wherein X_(a) is selected from W, P, R, V or L; and Z₍₁₋₆₎ is a stretchof any one to six amino acid residues. In certain embodiments ofpeptides having an amino acid sequence of either formula (a) or (b),Z₍₁₋₆₎ is one amino acid residue selected from G, L, F or M.

In certain aspects of the above embodiments of the invention, the BAFFspecific binding moiety comprises a CDR3 region which comprises apeptide selected from DWLLC (SEQ ID NO: 27), DPLLC (SEQ ID NO: 28),DRLLC (SEQ ID NO: 29), DLLLC (SEQ ID NO: 30), DLLFC (SEQ ID NO: 31),DPLFC (SEQ ID NO: 32), DPLMC (SEQ ID NO: 33), DPLTKEC (SEQ ID NO: 34),DLLTSSRRC (SEQ ID NO: 35), DPLLFPRDRC (SEQ ID NO: 36) orHGGRSTGLCGDVLLAGDV (SEQ ID NO: 37).

In certain other aspects of the above embodiments of the invention, theBAFF specific binding moiety comprises a CDR 3 region which comprises apeptide selected from RRARVIGGEYCRVQWQDV (SEQ ID NO: 38),LSNVHICCRFGSCADV (SEQ ID NO: 39), APTIISGCSIKRRDV (SEQ ID NO: 40), orTRYVVFSGSTCRMRRADV (SEQ ID NO: 41).

The present invention further provides a BAFF specific binding moietycomprising a CDR1 region comprising any one of the CDR1 peptidesequences shown in Table 1 in combination with a CDR3 region comprisingany one of the CDR3 peptides shown in Table 1, separated by a frameworkregion (see FW2-3 in Table 1), each as an independent embodiment of theinvention. In certain embodiments, the framework region interspersedbetween CDR1 and CDR3 comprises any one of the FW2-3 amino acidsequences shown in Table 1. The FW2-3 region in naturally-occurringVNARs is 53 amino acids in length, with insertions and deletions rarelyobserved. The FW2-3 region comprises hypervariable regions HV2 and HV4(see FIG. 1; and B. J. Fennell et al., J Mol Biol. 400 (2010) pp.155-170) which display some sequence variability and hence which can besuitable regions in which amino acid residues may be modified to createa variant of the BAFF specific binding moiety of the invention.

In any one of the individual embodiments described above, the BAFFspecific binding moiety may further comprise one or more of the FW1,FW2-3 or FW4 amino acid sequences shown in Table 1, in any functionalcombination. The present invention further provides a BAFF specificbinding moiety comprising one of the eighteen cloned peptide sequencesshown in Table 1.

Therefore, in one aspect, the invention provides a BAFF antagonistcompound comprising or consisting essentially of a VNAR derived BAFFspecific binding moiety which binds selectively to a BAFF polypeptide,preferably to human BAFF (Q9Y275-1) or to an epitope-containing fragmentof BAFF.

In one embodiment, a BAFF specific binding moiety or BAFF antagonistcompound of the invention binds to the target protein BAFF and decreasesor inhibits BAFF binding to BAFF-R. In other embodiments, the BAFFspecific binding moiety or BAFF antagonist compound inhibits BAFFinduced human B cell proliferation, and/or immunoglobulin production. Incertain embodiments, a BAFF specific binding moiety or BAFF antagonistcompound of the invention depletes B cells in vitro. In other certainembodiments, a BAFF specific binding moiety or BAFF antagonist compoundof the invention depletes B cells in vivo.

BAFF antagonist compound activity (“BAFF bioactivity”) may be determinedby one or more assays used to measure an activity which is eitherantagonism or agonism by an antibody. In certain embodiments, binding ofthe BAFF antagonist compound to BAFF is measured by a well-knownimmunoassay, such as for example an ELISA as described in Examples 2 and3. Any other binding assay which measures direct or indirect interactionof the BAFF antagonist compound to BAFF, or alternatively, which measurethe ability of a BAFFantagonist compound of the invention to compete forbinding to BAFF in the presence of a different BAFF binding compound(such as an anti-BAFF antibody) such as by a competitive inhibitionassay, may be used. Preferably, a selected assay measures the effect ofa BAFF antagonist compound of the invention on at least one biologicaleffect of native BAFF and in certain embodiments, compares the effect tothat of another native BAFF binding agent, e.g., to an anti-BAFFantibody. In vivo assays of BAFF bioactivity include, but are notlimited to: a mouse splenocyte assay such as that described in Example5; BAFF induced human B cell proliferation, IgGI production and/or humanB cell depleting activity.

Results of cellular in vitro assays may be further verified using one ormore in vivo animal models. A variety of accepted animal models ofimmune-related diseases or cancers may be used to characterize, e.g.,test the efficacy of, a BAFF antagonist compound or composition of theinvention. Animal models of immune-related diseases include bothnon-recombinant and recombinant (transgenic) animals. Tissue specificautoimmune diseases such as multiple sclerosis (MS) and experimentalautoimmune encephalomyelitis (EAE, a model for MS) in animal models maybe useful in characterizing BAFF antagonist compounds of the invention(see, e.g, Current Protocols in Immunology, unit 4.5). An acceptedanimal model for human autoimmune rheumatoid arthritis iscollagen-induced arthritis. Mouse and rat models are characterized bysynovitis, and erosion of cartilage and subchondral bone. BAFFantagonist compounds of the invention may be tested for activity againstautoimmune arthritis using such protocols (see, e.g, Current Protocolsin Immunology, unit 15.5; see also Issekutz, A. C. et al., Immunology,(1996) 88:569).

In one embodiment, the invention provides BAFF specific binding moietieswhich bind to a region of BAFF that interacts with BAFF-R. In a relatedembodiment, the invention provides BAFF specific binding moieties whichbind to a region of BAFF between amino acids 200 and 275 ofUniProtKB/Swiss-Prot: Q9Y275-1 (see also FIG. 4 of Gordon N C et al.,Biochemistry. 2003 May 27; 42(20):5977-83 for the BAFF/BLyS Receptor 3minimal TNF receptor-like module that encodes a highly focusedligand-binding site.)

According to another embodiment, a BAFF antagonist compound of theinvention binds to human BAFF in a standard ELISA or other similar assaywith an EC50 of 300 nM or less, and preferably 100 nM or less, 10 nM orless, or 1 nM or less. Thus, a BAFF antagonist compound of the inventionbinds to BAFF, e.g., hBAFF, in a standard ELISA or other similar assaywith an EC50 in a range of 0.1 nM to 300 nM, 0.5 nM to 300 nM, 1 nM to300 nM, 10 nM to 300 nM, 50 nM to 300 nM, 100 nM to 300 nM, 0.1 nM to100 nM, 0.5 nM to 100 nM, 1 nM to 100 nM, 5 nM to 100 nM, 10 nM to 100nM, 0.1 nM to 50 nM, 0.5 nM to 50 nM, 1 nM to 50 nM, 5 nM to 50 nM, 10nM to 50 nM.

According to another embodiment, a BAFF antagonist compound of theinvention competes with another antibody specific for binding to humanBAFF in a standard ELISA or other similar assay with an IC50 of 1micromolar or less, 500 nM or less, and preferably 100 nM or less, 50 nMor less, 25 nM or less, 10 nM or less, or 1 nM or less. Thus, a BAFFantagonist compound of the invention competes for binding to BAFF, e.g.,hBAFF, in a standard ELISA or other similar assay with an IC50 in arange of 0.1 nM to 1 micromolar, 1 nM to 1 micromolar, 10 nM to 1micromolar, 100 nM to 1 micromolar, 0.1 nM to 500 nM, 0.5 nM to 500 nM,1 nM to 500 nM, 10 nM to 500 nM, 50 nM to 500 nM, 100 nM to 500 nM, 250nM to 500 nM, 0.1 nM to 250 nM, 0.5 nM to 250 nM, 1 nM to 250 nM, 5 nMto 250 nM, 10 nM to 250 nM, 50 nM to 250 nM, 100 nM to 250 nM, 0.1 nM to100 nM, 0.5 nM to 100 nM, 1 nM to 100 nM, 5 nM to 100 nM, 10 nM to 100nM, 0.1 nM to 50 nM, 0.5 nM to 50 nM, 1 nM to 50 nM, or 10 nM to 50 nM.

According to another embodiment, a BAFF antagonist compound of theinvention depletes B cells in vitro with an EC₅₀ of 500 nM or less, 300nM or less, preferably 250 nM or less, 100 nM or less, 50 nM or less, 10nM or less, 1 nM or less or 100 pM or less. Thus, a BAFF antagonistcompound of the depletes B cells in vitro with an EC₅₀ in a range of 0.1nM to 500 nM, 0.5 nM to 500 nM, 1 nM to 500 nM, 10 nM to 500 nM, 50 nMto 500 nM, 100 nM to 500 nM, 250 nM to 500 nM, 0.1 nM to 250 nM, 0.5 nMto 250 nM, 1 nM to 250 nM, 5 nM to 250 nM, 10 nM to 250 nM, 50 nM to 250nM, 100 nM to 250 nM, 0.1 nM to 100 nM, 0.5 nM to 100 nM, 1 nM to 100nM, 5 nM to 100 nM, 10 nM to 100 nM, 0.1 nM to 50 nM, 0.5 nM to 50 nM, 1nM to 50 nM, or 10 nM to 50 nM.

According to another embodiment, a BAFF antagonist compound of theinvention inhibits B cell proliferation in a splenocyte proliferationassay or other similar in vitro cellular assay with an IC50 of 500 nM orless, 300 nM or less, preferably 250 nM or less, 100 nM or less, 50 nMor less, 10 nM or less, or 1 nM or less. Thus, a BAFF antagonistcompound of the inhibits B cell proliferation in a splenocyteproliferation assay or other similar in vitro cellular assay with anIC50 in a range of 0.1 nM to 500 nM, 0.5 nM to 500 nM, 1 nM to 500 nM,10 nM to 500 nM, 50 nM to 500 nM, 100 nM to 500 nM, 250 nM to 500 nM,0.1 nM to 250 nM, 0.5 nM to 250 nM, 1 nM to 250 nM, 5 nM to 250 nM, 10nM to 250 nM, 50 nM to 250 nM, 100 nM to 250 nM, 0.1 nM to 100 nM, 0.5nM to 100 nM, 1 nM to 100 nM, 5 nM to 100 nM, 10 nM to 100 nM, 0.1 nM to50 nM, 0.5 nM to 50 nM, 1 nM to 50 nM, or 10 nM to 50 nM.

The binding affinity, EC50 and IC50 ranges recited above are formeasurements on a BAFF specific VNAR monomer according to the presentinvention. Therapeutic versions of the invention include other molecularconfigurations, e.g., VNAR monomers fused to stabilizing heterologouspeptide regions, e.g., the Fc domain of an IgG or other immunoglobulinmolecule, which may be expressed and then further purified as multimers,such as covalent dimers. We envision that the activity of certain suchtherapeutic molecules will have even greater potency, preferably by atleast 2-10 fold higher potencies.

BAFF bioactivity may also or alternatively be measured by BAFF bindingaffinity, using any of a number of assays known in the art, such as asurface plasmon resonance assay (Example 5). According to anotherembodiment, a BAFF antagonist compound of the invention binds to humanBAFF in an affinity assay such as by surface plasmon resonance assaywith a binding affinity of 300 nM or less, and preferably 100 nM orless, 10 nM or less, 1 nM or less or 100 pM or less. Thus, a BAFFantagonist compound of the invention binds to BAFF, e.g., hBAFF, with anaffinity constant (K_(A)) in a range of 0.1 nM to 500 nM, 0.5 nM to 500nM, or 1 nM to 500 nM, 0.1 nM to 250 nM, 0.5 nM to 250 nM, or 1 nM to250 nM as measured, e.g., by surface plasmon resonance such as in aBIACore assay. In certain embodiments, a compound of the invention bindsto BAFF, e.g., hBAFF, with an affinity constant in a range of 0.1 nM to100 nM, 0.1 nM to 50 nM, or 0.1 nM to 10 nM, 0.5 nM to 100 nM, 0.5 nM to50 nM, or 0.5 nM to 10 nM, or 1 nM to 100 nM, 1 nM to 50 nM or 1 nM to10 nM, as measured, e.g., by surface plasmon resonance such as in aBIACore assay.

In another related embodiment, treatment of an accepted mouse model witha BAFF antagonist of the invention can reduce the percentage of B cellsin the blood or a tissue in vivo up to 70%, preferably up to 75%, morepreferably up to 80%, 90% or higher as compared to untreated controlanimals.

In some embodiments, BAFF antagonist compounds of the invention arespecific to human BAFF and do not measurably cross-react with othercross reactive ligands, such as with APRIL. In some embodiments, BAFFantagonist compounds of the invention are selective for binding to humanBAFF relative to cross reactive ligands, such as APRIL. In certainembodiments, the BAFF compound of the invention binds to hBAFF with a5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 500-fold or more higheraffinity compared to its binding affinity to a cross reactive ligand,such as APRIL. In some embodiments, a BAFF antagonist compound of theinvention is specific to human BAFF but also binds to or cross-reactswith one or more other mammalian BAFFs, e.g., with mouse BAFF(UniProtKB/Swiss-Prot: Q9WU72).

Pharmaceutically acceptable salts or solvates of any of the BAFFspecific binding compounds of the invention are likewise within thescope of the present invention.

As used herein, the term “pharmaceutically acceptable salt” refers to asalt that is not harmful to a patient or subject to which the salt inquestion is administered. It may be a salt chosen, e.g., among acidaddition salts and basic salts. Examples of acid addition salts includechloride salts, citrate salts and acetate salts. Examples of basic saltsinclude salts wherein the cation is selected from alkali metal cations,such as sodium or potassium ions, alkaline earth metal cations, such ascalcium or magnesium ions, as well as substituted ammonium ions, such asions of the type N(R1)(R2)(R3)(R4)+, wherein R1, R2, R3 and R4independently will typically designate hydrogen, optionally substitutedC₁₋₆-alkyl groups or optionally substituted C₂₋₆-alkenyl groups.Examples of relevant C₁₋₆-alkyl groups include methyl, ethyl, 1-propyland 2-propyl groups. Examples of C₂₋₆-alkenyl groups of possiblerelevance include ethenyl, 1-propenyl and 2-propenyl. Other examples ofpharmaceutically acceptable salts are described in “Remington'sPharmaceutical Sciences”, 17th edition, Alfonso R. Gennaro (Ed.), MarkPublishing Company, Easton, Pa., USA, 1985 (and more recent editionsthereof), in the “Encyclopaedia of Pharmaceutical Technology”, 3rdedition, James Swarbrick (Ed.), Informa Healthcare USA (Inc.), NY, USA,2007, and in J. Pharm. Sci. 66: 2 (1977).

The term “solvate” in the context of the present invention refers to acomplex of defined stoichiometry formed between a solute (in casu, apeptide compound or pharmaceutically acceptable salt thereof accordingto the invention) and a solvent. The solvent in this connection may, forexample, be water, ethanol or another pharmaceutically acceptable,typically small-molecular organic species, such as, but not limited to,acetic acid or lactic acid. When the solvent in question is water, sucha solvate is normally referred to as a hydrate.

In each of the sequences described above, and in each sequence describedherein, a C-terminal “—OH” moiety may be substituted for a C-terminal“—NH₂” moiety, and vice-versa.

Each of the specific compounds of the invention (e.g., BAFF bindingmoieties, BAFF antagonist peptides and compounds), and pharmaceuticallyacceptable salts and solvates thereof, constitutes an individualembodiment of the invention.

Derivatives, Variants, Conjugates

The invention further provides variants of a BAFF specific bindingmoiety of the invention, wherein the variant differs from the recitedamino acid sequence by up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more aminoacid residues (but by no more than that which retains 85%, 90%, 95%, 99%or more amino acid sequence identity) and retains BAFF bioactivity. BAFFbioactivity is measured by BAFF binding affinity, using any of a numberof assays know in the art. In certain embodiments, a compound of theinvention binds to BAFF, e.g., hBAFF, with an affinity constant in arange of 0.1 nM to 500 nM, 0.5 nM to 500 nM, or 1 nM to 500 nM, 0.1 nMto 250 nM, 0.5 nM to 250 nM, or 1 nM to 250 nM as measured, e.g., bysurface plasmon resonance such as in a BIACore assay. In certainembodiments, a compound of the invention binds to BAFF, e.g., hBAFF,with an affinity constant in a range of 0.1 nM to 100 nM, 0.1 nM to 50nM, or 0.1 nM to 10 nM, 0.5 nM to 100 nM, 0.5 nM to 50 nM, or 0.5 nM to10 nM, or 1 nM to 100 nM, 1 nM to 50 nM or 1 nM to 10 nM, as measured,e.g., by surface plasmon resonance such as in a BIACore assay. It willbe understood by one of skill in the art that amino acid residuesoutside of the conserved FW, CDR1 and CDR3 motifs are in general regionsin which amino acid modifications may be tolerated more readily withoutdeleteriously depleting BAFF binding activity.

A biologically active fragment of any of the foregoing sequences whichretains BAFF bioactivity is also encompassed by the present invention.Thus, in further aspects, the invention further comprises compoundshaving an amino acid sequence that is truncated (shortened), from the N-or C-terminus, relative to the full length sequence of compounds of theinvention. In some embodiments, the truncated compounds are truncated byup to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25 or more amino acid residues, counting from theC-terminus of a compound of the invention as disclosed above. Amino acidresidue outside of the conserved VNAR framework motifs are regions inwhich amino acid modifications may be better tolerated withoutdeleteriously depleting BAFF binding activity.

In some embodiments, the compounds of the invention may have at least40%, e.g., at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, 96%, 97%, 98%, or 99%, 99.5%, or 99.9% amino acid sequence identityto one of the BAFF antagonists disclosed herein, as long as the compoundretains a BAFF biological activity (as measured by BAFF bindingaffinity, EC50 or IC50) within a range described herein.

Thus in certain, BAFF specific binding compounds of the invention maycomprise the amino acid sequence of any one of the compounds shown inTable 1 (see below), or a functional variant thereof that has at leastabout 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 99.5% sequenceidentity to any one of the compounds in Table 1. A functional variant ofa polypeptide of the invention may inhibit at least one BAFF bioactivityby any one of the assays disclosed herein by at least 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5%, or 100%. Insome embodiments, a BAFF antagonist compound of the invention maycomprise one or more amino acid substitutions, e.g., conservative aminoacid substitutions, and retain BAFF binding activity of at least 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5%, or100% compared to the binding by an unmodified BAFF antagonist compoundof the invention, and/or compared to binding of any other availableanti-BAFF antibody, such as anti-human BAFF monoclonal antibodybelimumab.

Throughout the present specification, unless naturally occurring aminoacids are referred to by their full name (e.g. alanine, arginine, etc.),they are designated by their conventional three-letter or single-letterabbreviations (e.g. Ala or A for alanine, Arg or R for arginine, etc.).Unless otherwise indicated, reference is made to the L-isomeric forms ofthe amino acids in question. Where appropriate, the D-isomeric form ofan amino acid is indicated in the conventional manner by the prefix “D”before the conventional three-letter code (e.g. DAsp, DPhe).

In certain embodiments, the invention further provides a BAFF specificbinding moiety or BAFF antagonist comprising said binding moiety, inwhich there are one or more conservative amino acid substitutionsintroduced into the polypeptide sequence. As used herein, the term“conservative substitution” denotes that one or more amino acids arereplaced by another, biologically similar amino acid residue. Examplesinclude substitution of amino acid residues with similarcharacteristics, e. g. small amino acids, acidic amino acids, polaramino acids, basic amino acids, hydrophobic amino acids and aromaticamino acids. See, for example, the table below. An example of aconservative substitution with a residue normally not found inendogenous, mammalian peptides and proteins is the conservativesubstitution of Arg or Lys with, for example, ornithine, canavanine,aminoethylcysteine or another basic amino acid. For further informationconcerning phenotypically silent substitutions in peptides and proteins,see, e.g., Bowie et al., Science 247, 1306-1310, 1990. In the schemebelow are conservative substitutions of amino acids grouped byphysicochemical properties. I: neutral, hydrophilic, II: acids andamides, III: basic, IV: hydrophobic, V: aromatic, bulky amino acids.

I II III IV V A N H M F S D R L Y T E K I W P Q V G C

In some embodiments, a polypeptide of the invention may comprisefunctional fragments or variants of a BAFF specific binding moiety ofthe invention that have, at most, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or1 amino acid substitutions compared to a polypeptide sequence recitedherein, as long as it retains measurable biological activity alone or asa component of a BAFF antagonist compound. A polypeptide of theinvention may further be with or without a signal sequence. In certainembodiments, the retained activity is at least 50% that of the BAFFbinding moiety according to Table 1.

In some embodiments, a polypeptide of the invention shares at least 85%,90%, 95%, 96%, 97%, 98%, 99% or more amino acid sequence identity to anyone of the amino acid sequences of FW1, FW2-3, FW4, CDR1 or CDR3 ofTable 1, as long as it retains measurable biological activity alone oras a component of a BAFF antagonist compound. In certain embodiments,the retained activity is at least 50% that of the BAFF binding moietyaccording to Table 1.

BAFF specific VNAR comprising compounds of the invention may optionallybe conjugated to one or more additional agents which may includetherapeutic and/or diagnostic agents. Such agents include but are notlimited to chemotherapeutics such as cytostatic drugs, cytotoxins,radioisotopes, chelators, enzymes, nucleases, nucleic acids such as DNA,RNA or mixed nucleic acid oligonucleotides, including siRNAs, shRNAs,microRNAs, aptamers and the like; immunomodulators such as therapeuticantibodies, antibody and antibody-like fragments, inflammatory andanti-inflammatory cytokines, anti-inflammatory agents,radiotherapeutics, photoactive agents, diagnostic markers and the like

The invention further provides methods of making derivatives of BAFFspecific VNARs of the invention using biochemical engineering techniqueswell known to those of skill in the art. Such derivatives include, interalia, multivalent or multispecific molecules comprising a BAFF specificbinding moiety, including immunoconjugates. A large body of art isavailable relating to how to make and use antibody drug conjugates. Suchknowledge and skill in the art may be adapted for use with the BAFFspecific binding moieties and BAFF antagonist compounds of theinvention. See, e.g., WO2007/140371; WO2006/068867 specific to BAFF;methods relating to making and/or using different ligand antagonistconjugates may be applied. In certain embodiments, the BAFF selectivebinding moieties and antagonist compounds of the present inventioninclude covalently modified and conjugated polypeptides forms of thepolypeptides (e.g., immunoadhesins, radiolabeled or fluorescentlylabeled compounds, and the like). Methods for peptide conjugation andfor labeling polypeptides and conjugating molecules are well known inthe art.

Pharmaceutical Compositions

The present invention further provides pharmaceutical compositionscomprising a BAFF specific binding moiety or compound, or apharmaceutically acceptable salt or solvate thereof, according to theinvention, together with a pharmaceutically acceptable carrier,excipient or vehicle.

Accordingly, the present invention further provides a pharmaceuticalcomposition comprising a BAFF specific binding moiety or a BAFFantagonist compound comprising a BAFF specific binding moiety, as wellas variant and derivative compounds comprising a BAFF specific bindingmoiety of the invention. Certain embodiments of the pharmaceuticalcompositions of the invention are described in further detail below.

The present invention also provides pharmaceutical compositionscomprising a BAFF specific binding moiety or a BAFF antagonist compoundfor use in treating, ameliorating or preventing one or more diseases,conditions, disorders or symptoms relating to B cells and immunoglobulinproduction, as described in further detail below. Each such disease,condition, disorder or symptom is envisioned to be a separate embodimentwith respect to uses of a pharmaceutical composition according to theinvention.

Nucleic Acid Sequences That Encode a BAFF Selective Binding Moiety orBAFF Antagonist Compound

In one aspect, the invention provides an isolated nucleic acid whichencodes a BAFF specific binding moiety or BAFF antagonist compound ofthe invention, or a fragment or derivative thereof. The nucleic acid mayinclude, e.g., nucleic acid sequence encoding a polypeptide at least50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more, identicalto a polypeptide comprising one of the amino acid sequences of Table 1.The invention also provides an isolated nucleic acid molecule comprisinga sequence that hybridizes under stringent conditions to a nucleic acidsequence which encodes a BAFF specific binding moiety or BAFF antagonistcompound of the invention, or a fragment or derivative thereof, or theantisense or complement of any such sequence.

In another aspect, the invention provides an isolated nucleic acidmolecule encoding a fusion protein comprising at least two segments,wherein one of the segments comprises a polypeptide or fragment thereofhaving CDR 1, CDR3 or framework amino acid sequences shown in Table 1,and variants thereof according to the invention. In certain embodiments,a second segment comprises a heterologous signal polypeptide, aheterologous binding moiety, an immunoglobulin fragment such as a Fcdomain, or a detectable marker.

One aspect of the invention provides isolated nucleic acid moleculesthat encode BAFF specific binding moiety proteins or biologically activeportions thereof. Also included are nucleic acid fragments sufficientfor use as hybridization probes to identify BAFF binding moiety encodingnucleic acids and fragments for use as polymerase chain reaction (PCR)primers for the amplification or mutation of BAFF specific bindingmoiety encoding nucleic acid molecules.

As used herein, the term “nucleic acid molecule” is intended to includeDNA molecules, RNA molecules (e.g., mRNA, shRNA, siRNA, microRNA),analogs of the DNA or RNA generated using nucleotide analogs, andderivatives, fragments and homologs thereof. The nucleic acid moleculesof the invention may be single-, double-, or triple-stranded. A nucleicacid molecule of the present invention, e.g., a nucleic acid moleculeencoding any one of the amino acid sequences disclosed in Table 1, or acomplement of any of these nucleotide sequences, may be isolated usingsequence information provided herein and well known molecular biologicaltechniques (e.g., as described in Sambrook et al., Eds., MOLECULARCLONING: A LABORATORY MANUAL 2ND ED., Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1989; and Ausubel, et al., Eds.,CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York,N.Y., 1993).

A nucleic acid molecule of the invention may be amplified using any formof nucleic acid template and appropriate oligonucleotide primersaccording to standard PCR amplification techniques. Amplified nucleicacid may be cloned into an appropriate vector and characterized, e.g.,by restriction analysis or DNA sequencing. Furthermore, oligonucleotidescorresponding to nucleotide sequences that encode a BAFF selectivebinding moiety or BAFF antagonist compound of the invention may beprepared by standard synthetic techniques, e.g., using an automated DNAsynthesizer.

The term “oligonucleotide” as used herein refers to a series ofcovalently linked nucleotide (or nucleoside residues, includingribonucleoside or deoxyribonucleoside residues) wherein theoligonucleotide has a sufficient number of nucleotide bases to be usedin a PCR reaction. Oligonucleotides comprise portions of a nucleic acidsequence having at least about 10 nucleotides and as many as 50nucleotides, preferably about 15 nucleotides to 30 nucleotides.Oligonucleotides may be chemically synthesized and may be used asprobes. A short oligonucleotide sequence may be used to amplify,confirm, or reveal the presence of an identical, similar orcomplementary DNA or RNA in a particular cell or tissue.

Derivatives or analogs of the nucleic acid molecules (or proteins) ofthe invention include, inter alia, nucleic acid (or polypeptide)molecules having regions that are substantially homologous to thenucleic acid molecules or proteins of the invention, e.g., by at leastabout 45%, 50%, 70%, 80%, 95%, 98%, or even 99% identity (with apreferred identity of 80-99%) over a nucleic acid or amino acid sequenceof the same size or when compared to an aligned sequence in which thealignment is done by a computer homology program known in the art. Alsoincluded are nucleic acid molecules capable of hybridizing to thecomplement of a sequence encoding the proteins of the invention understringent or moderately stringent conditions (see, e.g., Ausubel, etal., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, NewYork, N.Y., 1993, and below). An exemplary program is the GAP program(Wisconsin Sequence Analysis Package, Version 8 for UNIX, GeneticsComputer Group, University Research Park, Madison, Wis.) using thedefault settings, which uses the algorithm of Smith and Waterman (1981)Adv. Appl. Math. 2:482489). Derivatives and analogs may be full lengthor other than full length, if the derivative or analog contains amodified nucleic acid or amino acid, as described below.

Stringent conditions are known to those skilled in the art and may befound in CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, N.Y.(1989), 6.3.1-6.3.6. In certain embodiments, stringent conditionstypically permit sequences at least about 65%, 70%, 75%, 85%, 90%, 95%,98%, or 99% homologous to each other to remain hybridized to each other.A non-limiting example of stringent hybridization conditions ishybridization in a high salt buffer comprising 6×SSC, 50 mM Tris-HCl (pH7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500 mg/mldenatured salmon sperm DNA at 65° C. This hybridization is followed byone or more washes in 0.2×SSC, 0.01% BSA at 50° C. The term “stringenthybridization conditions” as used herein refers to conditions underwhich a nucleic acid probe, primer or oligonucleotide will hybridize toits target sequence, but only negligibly or not at all to other nucleicacid sequences. Stringent conditions are sequence- and length-dependent,and depend on % (percent)-identity (or %-mismatch) over a certain lengthof nucleotide residues. Longer sequences hybridize specifically athigher temperatures than shorter sequences. Generally, stringentconditions are selected to be about 5° C. lower than the thermal meltingpoint (Tm) for the specific sequence at a defined ionic strength and pH.Stringent conditions may also be achieved with the addition ofdestabilizing agents, such as formamide.

Methods of Producing BAFF Specific VNAR Binding Moieties and BAFFAntagonists Comprising them.

The compounds of the invention may be manufactured by standard syntheticmethods, by use of recombinant expression systems, or by any othersuitable method. Thus, the compounds may be synthesized in a number ofways, including, e.g., methods comprising: (1) synthesizing apolypeptide or polypeptide component of a BAFF antagonist compound usingstandard solid-phase or liquid-phase methodology, either stepwise or byfragment assembly, and isolating and purifying the final peptidecompound product; (2) expressing a nucleic acid construct that encodes apolypeptide or polypeptide component of a BAFF antagonist compound in ahost cell and recovering the expression product from the host cell orhost cell culture; or (3) cell-free in vitro expression of a nucleicacid construct encoding a polypeptide or polypeptide component of a BAFFantagonist compound, and recovering the expression product; or by anycombination of the methods of (1), (2) or (3) to obtain fragments of thepeptide component, subsequently joining (e.g., ligating) the fragmentsto obtain the peptide component, and recovering the peptide component.

It may be preferable to synthesize a polypeptide or polypeptidecomponent of a BAFF antagonist compound of the invention by means ofsolid-phase or liquid-phase peptide synthesis. Compounds of theinvention may suitably be manufactured by standard synthetic methods.Thus, peptides may be synthesized by, e.g., methods comprisingsynthesizing the peptide by standard solid-phase or liquid-phasemethodology, either stepwise or by fragment assembly, and isolating andpurifying the final peptide product. In this context, reference may bemade to WO1998/11125 or, inter alia, Fields, G. B. et al., “Principlesand Practice of Solid-Phase Peptide Synthesis”; in: Synthetic Peptides,Gregory A. Grant (ed.), Oxford University Press (2nd edition, 2002) andthe synthesis examples herein.

Accordingly, the present invention also provides methods for producing aBAFF specific binding polypeptide of the invention according to aboverecited methods; a nucleic acid molecule encoding part or all of apolypeptide of the invention, a vector comprising at least one nucleicacid of the invention, expression vectors comprising at least onenucleic acid of the invention capable of producing a polypeptide of theinvention when introduced into a host cell, and a host cell comprising anucleic acid molecule, vector or expression vector of the invention.

BAFF antagonist compounds of the invention may be prepared usingrecombinant techniques well known in the art. In general, methods forproducing polypeptides by culturing host cells transformed ortransfected with a vector comprising the encoding nucleic acid andrecovering the polypeptide from cell culture are described in, e.g.,Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: ColdSpring Harbor Laboratory Press, 1989); Dieffenbach et al., PCR Primer: ALaboratory Manual (Cold Spring Harbor Laboratory Press, 1995).

A nucleic acid encoding a desired polypeptide may be inserted into areplication vector for further cloning (amplification) of the DNA or forexpression of the nucleic acid into RNA and protein. A multitude ofcloning and expression vectors are publicly available.

Expression vectors capable of directing transient or stable expressionof genes to which they are operably linked are well known in the art.The vector components generally include, but are not limited to, one ormore of the following: a heterologous signal sequence or peptide, anorigin of replication, one or more marker genes, an enhancer element, apromoter, and a transcription termination sequence, each of which iswell known in the art. Optional regulatory control sequences,integration sequences, and useful markers that can be employed are knownin the art.

Any suitable host cell may be used to produce BAFF antagonists of theinvention. Host cells may be cells stably or transiently transfected,transformed, transduced or infected with one or more expression vectorswhich drive expression of a polypeptide of the invention. Suitable hostcells for cloning or expressing nucleic acids of the invention includeprokaryote, yeast, or higher eukaryote cells. Eukaryotic microbes suchas filamentous fungi yeast, Arabidopsis, and other plant and animaleukaryotic host cells that may be grown in liquid culture are suitablecloning or expression hosts for vectors. Suitable host cells for theexpression of glycosylated polypeptides may also be derived frommulticellular organisms.

Creation and isolation of host cell lines producing a BAFF antagonistcompound of the invention can be accomplished using standard techniquesknown in the art. Mammalian cells are preferred host cells forexpression of peptide antagonists. Particularly useful mammalian cellsinclude, inter alia, HEK 293, NSO, DG-44, and CHO cells, but any othersuitable host cell may be used according to the invention. Preferably,the BAFF antagonist compounds are secreted into the medium in which thehost cells are cultured, from which the BAFF antagonist compounds may berecovered or purified.

When a polypeptide is produced in a recombinant cell other than one ofhuman origin, it is typically free of polypeptides of human origin. Incertain embodiments, it is advantageous to separate a polypeptide awayfrom other recombinant cell components such as host cell polypeptides toobtain preparations that are of high purity or substantiallyhomogeneous. As a first step, culture medium or cell lysates may becentrifuged to remove particulate cell debris and suitable proteinpurification procedures may be performed. Such procedures include, interalia, fractionation (e.g., size separation by gel filtration or chargeseparation by ion-exchange column); ethanol precipitation; Protein ASepharose columns to remove contaminants such as IgG; hydrophobicinteraction chromatography; reverse phase HPLC; chromatography on silicaor on cation-exchange resins such as DEAE and the like;chromatofocusing; electrophoretic separations; ammonium sulfateprecipitation; gel filtration using, for example, Sephadex beads such asG-75. Any number of biochemical purification techniques may be used toincrease the purity of a BAFF antagonist compound of the invention.

Methods of Detection

In certain embodiments, the BAFF antagonist compounds of the inventionmay be used to detect and quantify levels of BAFF, or cells that expressBAFF-R. This can be achieved, for example, by contacting a test sample(such as an in vitro sample) and a control sample with a BAFF specificbinding moiety of the invention, or a BAFF antagonist compoundcomprising it, under conditions which permit formation of a complexbetween the antagonist and BAFF, or between BAFF and BAFF-R, or both.Any bound BAFF complexes are detected and/or quantified in BAFF specificVNAR containing samples and control samples.

Accordingly, the invention further provides methods for detecting thepresence of BAFF or BAFF-R in a sample, or measuring the amount ofeither of the foregoing, comprising contacting the sample, andpreferably a control sample, with a BAFF antagonist compound of theinvention under conditions that permit complex formation between theBAFF binding moiety of the antagonist and BAFF, e.g., human BAFF.Formation or inhibition of formation of a BAFF/BAFF-R complex is thendetected and/or quantified. A variety of tests can be designed based onfeatures of binding or competition for binding. For example, thepresence of BAFF-R in a test sample may be detected directly, or may bedetected and quantified based on the ability to compete for binding ofBAFF-R by BAFF. In general, the difference in complex formation betweena test sample and a control sample is indicative of a bindinginteraction.

Kits for Detecting or Quantifying BAFF in a Sample

Also within the scope of the invention are kits comprising at least oneBAFF specific binding moiety or BAFF antagonist compound or compositionof the invention, and optionally, instructions for use. Kits may beuseful for quantifying BAFF or BAFF-R in a sample, or may be useful fordetection of BAFF, such as in diagnostics methods. The kit may furtheror alternatively comprise at least one nucleic acid encoding a BAFFspecific binding moiety of the invention. A kit of the invention mayoptionally comprise at least one additional reagent (e.g., standards,markers and the like). Kits typically include a label indicating theintended use of the contents of the kit. The kit may further comprisereagents and other tools for measuring BAFF in a sample or in a subject,or for diagnosing whether a patient belongs to a group that responds toa BAFF antagonist which makes use of a compound, composition or relatedmethod of the invention as described herein.

BAFF Antagonists and Compositions for Use in Methods of MedicalTreatment

The present invention provides a BAFF antagonist compound for use, aloneor in combination with one or more additional therapeutic agents in apharmaceutical composition, for treatment or prophylaxis of conditions,diseases and disorders responsive to modulation (such as inhibiting orblocking) of the interaction between BAFF-R and BAFF.

BAFF antagonist compounds and pharmaceutical compositions of theinvention may be used in the treatment of a variety of conditions,disorders or diseases involving B cells. Treatment of with a BAFFantagonist compound of the invention preferably leads to in vivo B celldepletion. As such, BAFF antagonist compounds of the invention, andcompositions comprising them, will be useful in methods for treating avariety of pathological disorders in which killing or depleting B cellsmay be beneficial, such as, inter alia, systemic lupus erythematosus,rheumatoid arthritis, multiple sclerosis and certain other autoimmunedisorders or diseases, and e.g., lymphomas, leukemias and myelomas. Thepresent invention provides methods for suppressing B cell proliferation,and treating B cell disorders, including neoplasms, tumors and othermalignancies, comprising the step of administering to a subject in needthereof a therapeutically effective amount of a BAFF antagonistcompound.

In general, B-cell disorders may be classified as defects of B celldevelopment and/or immunoglobulin production (immunodeficiencies) and/orexcessive or uncontrolled B cell proliferation (e.g., in leukemias,lymphomas and myelomas). As used herein, a “B cell disorder” is intendedto refer to both types of disease, unless otherwise indicated bycontext.

The invention thus provides methods of treatment or prevention of a Bcell associated disorder, the method comprising the step ofadministering to a subject (e.g., a patient) in need thereof atherapeutically effective amount of the BAFF antagonist compound orpharmaceutical composition comprising a BAFF antagonist compound of theinvention, as described herein. As used herein, an “effective amount,” a“therapeutically effective amount” or an “effective dose” is an amountof a composition (e.g., a therapeutic composition or agent) thatproduces at least one desired therapeutic effect in a subject, such aspreventing or treating a target condition or beneficially alleviating asymptom associated with the condition.

The most desirable therapeutically effective amount is an amount thatwill produce a desired efficacy of a particular treatment selected byone of skill in the art for a given subject in need thereof. This amountwill vary depending upon a variety of factors understood by the skilledworker, including but not limited to the characteristics of thetherapeutic compound (including activity, pharmacokinetics,pharmacodynamics, and bioavailability), the physiological condition ofthe subject (including age, sex, disease type and stage, generalphysical condition, responsiveness to a given dosage, and type ofmedication), the nature of the pharmaceutically acceptable carrier orcarriers in the formulation, and the route of administration. Oneskilled in the clinical and pharmacological arts will be able todetermine a therapeutically effective amount through routineexperimentation, namely by monitoring a subject's response toadministration of a compound and adjusting the dosage accordingly. See,e.g., Remington: The Science and Practice of Pharmacy 21st Ed., Univ. ofSciences in Philadelphia (USIP), Lippincott Williams & Wilkins,Philadelphia, Pa., 2005.

In certain embodiments, BAFF antagonists and pharmaceutical compositionsof the invention may be used to treat or prevent inflammatoryconditions, including but not limited to rheumatoid arthritis (RA),multiple sclerosis (MS), psoriasis, Type 1 diabetes mellitus,scleroderma/systemic sclerosis, Sjögren's syndrome (SS), systemic lupuserythematosus (SLE), certain forms of thyroiditis, certain forms ofuveitis, vitiligo, granulomatosis with polyangiitis (Wegener's) alopeciaareata, autoimmune hemolytic anemia, autoimmune hepatitis,dermatomyositis, certain forms of juvenile idiopathic arthritis, (acute)glomerulonephritis, Graves' disease, Guillain-Barré syndrome, idiopathicthrombocytopenic purpura (ITP), myasthenia gravis, some forms ofmyocarditis, pemphigus/pemphigoid, pernicious anemia, polyarteritisnodosa, polymyositis, primary biliary cirrhosis.

In certain embodiments, the present invention provides methods fortreating B cell malignancies or neoplasms and other blood cellassociated cancers in a mammalian subject, such as a human, the methodcomprising the step of administering to a subject in need thereof atherapeutically effective amount of a BAFF antagonist compound orpharmaceutical composition comprising a BAFF antagonist compound. Insome embodiments, a BAFF antagonist compound of the invention is used totreat a B cell-, plasma cell- or antibody-mediated disease or disorder,such as for example Multiple Myeloma (MM), chronic lymphocytic leukemia(CLL), non-secretory multiple myeloma, Smoldering multiple myeloma,Monoclonal gammopathy of undetermined significance (MGUS), Solitaryplasmacytoma (Bone, Extramedullar), Lymphoplasmacytic lymphoma (LPL),Waldenstrom's Macroglobulinemia, Plasma cell leukemia, PrimaryAmyloidosis (AL), Heavy chain disease, POEMS syndrome/osteoscleroticmyeloma, Type I and II cryoglobulinemia, Light chain deposition disease,Goodpasture's syndrome, Pemphigus and Pemphigoid disorders, andEpidermolysis bullosa acquisita; or any Non-Hodgkin's Lymphoma B-cellleukemia or Hodgkin's lymphoma (HL) with BAFF-R expression or anyconditions associated with the development of neutralizing antibodies torecombinant protein replacement therapy.

BAFF antagonist compounds and compositions of the invention may bebeneficial in treating or diagnosing certain B-cell neoplasms. B cellmalignancies or neoplasms that may be diagnosed or treated using themethods described herein include, but are not limited to, Non-Hodgkin'sLymphomas (NHL), Diffuse Large B Cell Lymphoma (DLBCL), Smalllymphocytic lymphoma (SLL/CLL), Lymphoplasmacytoid lymphoma, Mantle celllymphoma (MCL), Follicular lymphoma (FL), Marginal zone lymphoma (MZL),Extranodal mucosa-associated lymphoid tissue lymphoma (MALT lymphoma),Nodal (Monocytoid B-cell lymphoma), Splenic, Diffuse large celllymphoma, Burkitt's lymphoma, Lymphoblastic lymphoma, precursorB-lymphoblastic leukemia, B-cell chronic lymphocytic leukemia, SolitaryPlasmacytoma (Bone, Extramedullar), Multiple Myeloma (MM), andSmoldering Multiple Myeloma (SMM). Treatment and diagnosis of otherB-cell neoplasms are also considered to fall within the scope of thepresent invention.

In certain embodiments, an autoimmune disease or cancer to be treatedusing a BAFF antagonist compound or composition according to theinvention is selected from (a) Systemic Lupus Erythematosus; (b)Rheumatoid Arthritis; (c) Multiple Sclerosis; (d) IdiopathicThrombocytopenic Purpura; (e) Sjogren's syndrome; (f) Diabetes; (g)Waldenstrom's macroglobulinaemia; (h) acute lymphocytic leukemia; (i)chronic lymphocytic leukemia; (j) non-Hodgkin's lymphoma; (k) multiplemyeloma; (l) vasculitis; and (m) graft or transplant rejection.

In certain embodiments, the condition, disease or disorder is selectedfrom Multiple Myeloma (MM), Smoldering Multiple Myeloma (SMM), ChronicLymphocytic Leukemia (CLL), Solitary Plasmacytoma (Bone, Extramedullar),Waldenstrom's Macroglobulinemia and Idiopathic thrombocytopenic purpura(ITP).

The BAFF antagonist compounds and related compositions of the inventionmay be used in the manufacture of a pharmaceutical composition ormedicament for the treatment of one or more of each of the B cellrelated conditions, diseases and disorders described herein.

Formulations, Administration and Dosing

BAFF antagonist compounds of the present invention, or salts thereof,may be formulated as pharmaceutical compositions prepared for storage oradministration, which typically comprise a therapeutically effectiveamount of a compound of the invention, or a salt thereof, in apharmaceutically acceptable carrier.

The therapeutically effective amount of a compound of the presentinvention will depend on the route of administration, the type of mammalbeing treated, and the physical characteristics of the specific mammalunder consideration. These factors and their relationship to determiningthis amount are well known to skilled practitioners in the medical arts.This amount and the method of administration can be tailored to achieveoptimal efficacy, and may depend on such factors as weight, diet,concurrent medication and other factors, well known to those skilled inthe medical arts. The dosage sizes and dosing regimen most appropriatefor human use may be guided by the results obtained by the presentinvention and may be confirmed in properly designed clinical trials.

An effective dosage and treatment protocol may be determined byconventional means, starting with a low dose in laboratory animals andthen increasing the dosage while monitoring the effects, andsystematically varying the dosage regimen as well. Numerous factors maybe taken into consideration by a clinician when determining an optimaldosage for a given subject. Such considerations are known to the skilledperson. The term “pharmaceutically acceptable carrier” includes any ofthe standard pharmaceutical carriers. Pharmaceutically acceptablecarriers for therapeutic use are well known in the pharmaceutical art,and are described, for example, in Remington's Pharmaceutical Sciences,Mack Publishing Co. (A. R. Gennaro edit. 1985). For example, sterilesaline and phosphate-buffered saline at slightly acidic or physiologicalpH may be used. Exemplary pH buffering agents include phosphate,citrate, acetate, tris/hydroxymethyl)aminomethane (TRIS),N-Tris(hydroxymethyl)methyl-3-aminopropanesulphonic acid (TAPS),ammonium bicarbonate, diethanolamine, histidine, which is a preferredbuffer, arginine, lysine, or acetate or mixtures thereof. The termfurther encompasses any agents listed in the US Pharmacopeia for use inanimals, including humans.

The term “pharmaceutically acceptable salt” refers to the salt of thecompounds. Salts include pharmaceutically acceptable salts such as acidaddition salts and basic salts. Examples of acid addition salts includehydrochloride salts, citrate salts and acetate salts. Examples of basicsalts include salts where the cation is selected from alkali metals,such as sodium and potassium, alkaline earth metals such as calcium, andammonium ions ⁺N(R³)₃(R⁴), where R³ and R⁴ independently designateoptionally substituted C₁₋₆-alkyl, optionally substituted C₂₋₆-alkenyl,optionally substituted aryl, or optionally substituted heteroaryl. Otherexamples of pharmaceutically acceptable salts are described in“Remington's Pharmaceutical Sciences”, 17th edition. Ed. Alfonso R.Gennaro (Ed.), Mark Publishing Company, Easton, Pa., U.S.A., 1985 andmore recent editions, and in the Encyclopaedia of PharmaceuticalTechnology.

“Treatment” is an approach for obtaining beneficial or desired clinicalresults. For the purposes of this invention, beneficial or desiredclinical results include, but are not limited to, alleviation ofsymptoms, diminishment of extent of disease, stabilized (i.e., notworsening) state of disease, delay or slowing of disease progression,amelioration or palliation of the disease state, and remission (whetherpartial or total), whether detectable or undetectable. “Treatment” canalso mean prolonging survival as compared to expected survival if notreceiving treatment. “Treatment” is an intervention performed with theintention of preventing the development or altering the pathology of adisorder. Accordingly, “treatment” refers to both therapeutic treatmentand prophylactic or preventative measures in certain embodiments. Thosein need of treatment include those already with the disorder as well asthose in which the disorder is to be prevented. By treatment is meantinhibiting or reducing an increase in pathology or symptoms whencompared to the absence of treatment, and is not necessarily meant toimply complete cessation of the relevant condition.

The pharmaceutical compositions can be in unit dosage form. In suchform, the composition is divided into unit doses containing appropriatequantities of the active component. The unit dosage form can be apackaged preparation, the package containing discrete quantities of thepreparations, for example, packeted tablets, capsules, and powders invials or ampoules. The unit dosage form can also be a capsule, cachet,or tablet itself, or it can be the appropriate number of any of thesepackaged forms. It may be provided in single dose injectable form, forexample in the form of a pen. Compositions may be formulated for anysuitable route and means of administration.

Pharmaceutically acceptable carriers or diluents include those used informulations suitable for oral, rectal, nasal or parenteral (includingsubcutaneous, intramuscular, intravenous, intradermal, and transdermal)administration. The formulations may conveniently be presented in unitdosage form and may be prepared by any of the methods well known in theart of pharmacy. Subcutaneous or transdermal modes of administration maybe particularly suitable for the compounds described herein.

An acceptable route of administration may refer to any administrationpathway known in the art, including but not limited to aerosol, enteral,nasal, ophthalmic, oral, parenteral, rectal, vaginal, or transdermal(e.g., topical administration of a cream, gel or ointment, or by meansof a transdermal patch). “Parenteral administration” is typicallyassociated with injection at or in communication with the intended siteof action, including infraorbital, infusion, intraarterial,intracapsular, intracardiac, intradermal, intramuscular,intraperitoneal, intrapulmonary, intraspinal, intrasternal, intrathecal,intrauterine, intravenous, subarachnoid, subcapsular, subcutaneous,transmucosal, or transtracheal administration.

In another aspect, the present invention provides a composition, e.g., apharmaceutical composition, comprising one or a combination of differentBAFF antagonist compounds of the invention, or a VNAR sequencecontaining, BAFF specific binding region thereof, or an ester, salt oramide of any of the foregoing, and at least one pharmaceuticallyacceptable carrier. Such compositions may include one or more differentBAFF specific binding moieties or compounds in combination to produce animmunoconjugate or multispecific molecule comprising at least one BAFFspecific binding moiety. For example, a pharmaceutical composition ofthe invention may comprise a combination of BAFF specific bindingmoieties which bind to different epitopes of BAFF or which otherwisehave complementary biological activities.

Pharmaceutical compositions of the invention may be administered aloneor in combination with one or more other therapeutic or diagnosticagents. A combination therapy may include a BAFF antagonist compound ofthe present invention combined with at least one other therapeutic agentselected based on the particular patient, disease or condition to betreated. Examples of other such agents include, inter alia, a cytotoxic,anti-cancer or chemotherapeutic agent, an anti-inflammatory oranti-proliferative agent, an antimicrobial or antiviral agent, growthfactors, cytokines, an analgesic, a therapeutically active smallmolecule or polypeptide, a single chain antibody, a classical antibodyor fragment thereof, or a nucleic acid molecule which modulates one ormore signaling pathways, and similar modulating therapeutics which maycomplement or otherwise be beneficial in a therapeutic or prophylactictreatment regimen.

As used herein, “pharmaceutically acceptable carrier” includes any andall physiologically acceptable, i.e., compatible, solvents, dispersionmedia, coatings, antimicrobial agents, isotonic and absorption delayingagents, and the like. In certain embodiments, the carrier is suitablefor intravenous, intramuscular, subcutaneous, parenteral, spinal orepidermal administration (e.g., by injection or infusion). Depending onselected route of administration, the BAFF specific binding moietycomprising compound or component may be coated in a material ormaterials intended to protect the compound from the action of acids andother natural inactivating conditions to which the active BAFF bindingmoiety may encounter when administered to a subject by a particularroute of administration.

As above, a compound of the invention may encompass one or morepharmaceutically acceptable salts. As used herein a “pharmaceuticallyacceptable salt” retains qualitatively a desired biological activity ofthe parent compound without imparting any undesired effects relative tothe compound. Examples of pharmaceutically acceptable salts include acidaddition salts and base addition salts. Acid addition salts includesalts derived from nontoxic inorganic acids, such as hydrochloric,nitric, phosphorous, phosphoric, sulfuric, hydrobromic, hydroiodic andthe like, or from nontoxic organic acids such as aliphatic mono- anddi-carboxylic acids, phenyl-substituted alkanoic acids, hydroxy alkanoicacids, aromatic acids, aliphatic and aromatic sulfonic acids and thelike. Base addition salts include salts derived from alkaline earthmetals, such as sodium, potassium, magnesium, calcium and the like, aswell as from nontoxic organic amines, such as N,N′-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline,diethanolamine, ethylenediamine, procaine and the like.

A pharmaceutical composition of the invention also optionally includes apharmaceutically acceptable antioxidant. Exemplary pharmaceuticallyacceptable antioxidants are water soluble antioxidants such as ascorbicacid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite,sodium sulfite and the like; oil-soluble antioxidants, such as ascorbylpalmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene(BHT), lecithin, propylgallate, alpha-tocopherol, and the like; andmetal chelating agents, such as citric acid, ethylenediamine tetraaceticacid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

Examples of suitable aqueous and nonaqueous carriers that may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyloleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

BAFF antagonist compositions may also contain adjuvants such aspreservatives, wetting agents, emulsifying agents and dispersing agents.Prevention of presence of microorganisms may be ensured both bysterilization procedures, and by the inclusion of various antibacterialand antifungal agents, for example, paraben, chlorobutanol, phenolsorbic acid, and the like. Isotonic agents, such as sugars, sodiumchloride, and the like into the compositions, may also be desirable. Inaddition, prolonged absorption of the injectable pharmaceutical form maybe brought about by the inclusion of agents which delay absorption suchas, aluminum monostearate and gelatin.

Exemplary pharmaceutically acceptable carriers include sterile aqueoussolutions or dispersions and sterile powders for the extemporaneouspreparation of sterile injectable solutions or dispersion. Such mediaand reagents for pharmaceutically active substances are known in theart. The pharmaceutical compositions of the invention may include anyconventional media or agent unless any is incompatible with the activeBAFF antagonist compound. Supplementary active compounds may further beincorporated into the compositions.

Therapeutic compositions are typically sterile and stable under theconditions of manufacture and storage. The composition may be formulatedas a solution, microemulsion, liposome, or other ordered structuresuitable to high drug concentration. The carrier may be a solvent ordispersion medium containing, for example, water, alcohol such asethanol, polyol (e.g., glycerol, propylene glycol, and liquidpolyethylene glycol), or any suitable mixtures. The proper fluidity maybe maintained, for example, by the use of a coating such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by use of surfactants according to formulation chemistry well knownin the art. In certain embodiments, isotonic agents, e.g., sugars,polyalcohols such as mannitol, sorbitol, or sodium chloride may bedesirable in the composition. Prolonged absorption of injectablecompositions may be brought about by including in the composition anagent that delays absorption for example, monostearate salts andgelatin.

Solutions or suspensions used for intradermal or subcutaneousapplication typically include one or more of: a sterile diluent such aswater for injection, saline solution, fixed oils, polyethylene glycols,glycerine, propylene glycol or other synthetic solvents; antibacterialagents such as benzyl alcohol or methyl parabens; antioxidants such asascorbic acid or sodium bisulfite; chelating agents such asethylenediaminetetraacetic acid; buffers such as acetates, citrates orphosphates; and tonicity adjusting agents such as, e.g., sodium chlorideor dextrose. The pH can be adjusted with acids or bases, such ashydrochloric acid or sodium hydroxide, or buffers with citrate,phosphate, acetate and the like. Such preparations may be enclosed inampoules, disposable syringes or multiple dose vials made of glass orplastic.

Sterile injectable solutions may be prepared by incorporating a BAFFspecific binding moiety (or a BAFF antagonist compound comprising such amoiety) in the required amount in an appropriate solvent with one or acombination of ingredients described above, as required, followed bysterilization microfiltration. Dispersions may be prepared byincorporating the active compound into a sterile vehicle that contains adispersion medium and other ingredients, such as those described above.In the case of sterile powders for the preparation of sterile injectablesolutions, the methods of preparation are vacuum drying andfreeze-drying (lyophilization) that yield a powder of the activeingredient in addition to any additional desired ingredient from asterile-filtered solution thereof.

When a therapeutically effective amount of a BAFF antagonist compound ofthe invention is administered by, e.g., intravenous, cutaneous orsubcutaneous injection, the binding agent will be in the form of apyrogen-free, parenterally acceptable aqueous solution. Methods forpreparing parenterally acceptable protein solutions, taking intoconsideration appropriate pH, isotonicity, stability, and the like, arewithin the skill in the art. A preferred pharmaceutical composition forintravenous, cutaneous, or subcutaneous injection will contain, inaddition to binding agents, an isotonic vehicle such as sodium chlorideinjection, Ringer's injection, dextrose injection, dextrose and sodiumchloride injection, lactated Ringer's injection, or other vehicle asknown in the art. A pharmaceutical composition of the present inventionmay also contain stabilizers, preservatives, buffers, antioxidants, orother additives well known to those of skill in the art.

The amount of active ingredient which can be combined with a carriermaterial to produce a single dosage form will vary depending on avariety of factors, including the subject being treated, and theparticular mode of administration. In general, it will be an amount ofthe composition that produces an appropriate therapeutic effect underthe particular circumstances. Generally, out of one hundred percent,this amount will range from about 0.01 percent to about ninety-ninepercent of active ingredient, from about 0.1 percent to about 70percent, or from about 1 percent to about 30 percent of activeingredient in combination with a pharmaceutically acceptable carrier.

Dosage regimens may be adjusted to provide the optimum desired response(e.g., a therapeutic response). For example, a single bolus may beadministered, several divided doses may be administered over time, orthe dose may be proportionally reduced or increased as indicated by theparticular circumstances of the therapeutic situation, on a case by casebasis. It is especially advantageous to formulate parenteralcompositions in dosage unit forms for ease of administration anduniformity of dosage when administered to the subject or patient. Asused herein, a dosage unit form refers to physically discrete unitssuitable as unitary dosages for the subjects to be treated; each unitcontaining a predetermined quantity of active compound calculated toproduce a desired therapeutic effect in association with the requiredpharmaceutical carrier. The specification for the dosage unit forms ofthe invention depend on the specific characteristics of the activecompound and the particular therapeutic effect(s) to be achieved, takinginto consideration and the treatment and sensitivity of any individualpatient.

For administration of a BAFF antagonist compound, the dosage range willgenerally be from about 0.0001 to 100 mg/kg, and more usually 0.01 to 5mg/kg, of the host body weight. Exemplary dosages may be 0.25 mg/kg bodyweight, 1 mg/kg body weight, 3 mg/kg body weight, 5 mg/kg body weight or10 mg/kg body weight or within the range of 1-10 mg/kg. An exemplarytreatment regime is a once or twice daily administration, or a once ortwice weekly administration, once every two weeks, once every threeweeks, once every four weeks, once a month, once every two or threemonths or once every three to 6 months. Dosages may be selected andreadjusted by the skilled health care professional as required tomaximize therapeutic benefit for a particular subject, e.g., patient.BAFF antagonist compounds will typically be administered on multipleoccasions. Intervals between single dosages can be, for example, 2-5days, weekly, monthly, every two or three months, every six months, oryearly. Intervals between administrations can also be irregular, basedon regulating blood levels of BAFF antagonist to the target BAFF ligandin the subject or patient. In some methods, dosage is adjusted toachieve a plasma antagonist concentration of about 1-1000 μg/ml and insome methods about 25-300 μg/ml. Dosage regimens for a BAFF antagonistof the invention include intravenous administration of 1 mg/kg bodyweight or 3 mg/kg body weight with the compound administered every twoto four weeks for six dosages, then every three months at 3 mg/kg bodyweight or 1 mg/kg body weight.

In certain embodiments, two or more BAFF antagonist compounds withdifferent binding properties may be administered simultaneously orsequentially, in which case the dosage of each administered antagonistmay be adjusted to fall within the ranges described herein.

In certain embodiments, a BAFF antagonist compound of the invention maybe administered as a sustained release formulation, in which case lessfrequent administration is required. Dosage and frequency vary dependingon the half-life of the BAFF antagonist in the subject or patient. Thedosage and frequency of administration may vary depending on whether thetreatment is therapeutic or prophylactic (e.g., preventative), and maybe adjusted during the course of treatment. In certain prophylacticapplications, a relatively low dosage is administered at relativelyinfrequent intervals over a relatively long period of time. Somesubjects may continue to receive treatment over their lifetime. Incertain therapeutic applications, a relatively high dosage at relativelyshort intervals is sometimes required until progression of the diseaseis reduced or until the patient shows partial or complete ameliorationof symptoms of disease. Thereafter, the patient may be switched to asuitable prophylactic dosing regimen.

Actual dosage levels of the BAFF antagonist compound alone or incombination with one or more other active ingredients in thepharmaceutical compositions of the present invention may be varied so asto obtain an amount of the active ingredient which is effective toachieve the desired therapeutic response for a particular patient,composition, and mode of administration, without causing deleteriousside effects to the subject or patient. A selected dosage level willdepend upon a variety of factors, such as pharmacokinetic factors,including the activity of the particular BAFF antagonist compound orcomposition employed, or the ester, salt or amide thereof, the route ofadministration, the time of administration, the rate of excretion of theparticular compound being employed, the duration of the treatment, otherdrugs, compounds and/or materials used in combination with theparticular compositions employed, the age, sex, weight, condition,general health and prior medical history of the subject or patient beingtreated, and similar factors well known in the medical arts.

Administration of a “therapeutically effective dosage” of a BAFFantagonist compound of the invention may result in a decrease inseverity of disease symptoms, an increase in frequency and duration ofdisease symptom-free periods, or a prevention of impairment ordisability due to the disease affliction.

A BAFF antagonist compound or composition of the present invention maybe administered via one or more routes of administration, using one ormore of a variety of methods known in the art. As will be appreciated bythe skilled worker, the route and/or mode of administration will varydepending upon the desired results. Routes of administration for BAFFantagonist compounds or compositions of the invention include, e.g.,intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous,spinal or other parenteral routes of administration, for example byinjection or infusion. The phrase “parenteral administration” as usedherein refers to modes of administration other than enteral and topicaladministration, usually by injection, and includes, without limitation,intravenous, intramuscular, intraarterial, intrathecal, intracapsular,intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal,subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid,intraspinal, epidural and intrastemal injection and infusion.

In other embodiments, a BAFF antagonist compound or composition of theinvention may be administered by a non-parenteral route, such as atopical, epidermal or mucosal route of administration, for example,intranasally, orally, vaginally, rectally, sublingually or topically.

As described elsewhere herein, an active BAFF antagonist compound may beprepared with carriers that will protect the compound against rapidrelease, such as a controlled release formulation, including implants,transdermal patches, and microencapsulated delivery systems.Biodegradable, biocompatible polymers can be used, such as ethylenevinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, and polylactic acid. Many methods for the preparationof such formulations are patented or generally known to those skilled inthe art. See, e.g., Sustained and Controlled Release Drug DeliverySystems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.

Therapeutic compounds or compositions of the invention may beadministered with one or more of a variety of medical devices known inthe art. For example, in one embodiment, a therapeutic BAFF antagonistcomposition of the invention may be administered with a needlelesshypodermic injection device. Examples of well-known implants and modulesuseful in the present invention are in the art, including e.g.,implantable micro-infusion pumps for controlled rate delivery; devicesfor administering through the skin; infusion pumps for delivery at aprecise infusion rate; variable flow implantable infusion devices forcontinuous drug delivery; and osmotic drug delivery systems. These andother such implants, delivery systems, and modules are known to thoseskilled in the art.

In certain embodiments, the BAFF antagonist compound or composition ofthe invention may be formulated to ensure a desired distribution invivo. For example, the blood-brain barrier (BBB) excludes many highlyhydrophilic compounds. To target a therapeutic compound or compositionof the invention to a particular in vivo location, they can beformulated, for example, in liposomes which may comprise one or moremoieties that are selectively transported into specific cells or organs,thus enhancing targeted drug delivery. Exemplary targeting moietiesinclude folate or biotin; mannosides; antibodies; surfactant protein Areceptor; p120 and the like.

Delivery Devices and Kits

In certain embodiments, the invention relates to a device comprising oneor more BAFF antagonist compounds of the invention, or pharmaceuticallyacceptable salts or solvates thereof, for delivery to a subject. Thus,one or more compounds of the invention or pharmaceutically acceptablesalts or solvates thereof can be administered to a patient in accordancewith the present invention via a variety of delivery methods, including:intravenous, subcutaneous, intramuscular or intraperitoneal injection;oral administration; transdermal administration; pulmonary ortransmucosal administration; administration by implant, osmotic pump,cartridge or micro pump; or by other means recognized by a person ofskill in the art.

In some embodiments, the invention relates to a kit comprising one ormore peptides, or pharmaceutically acceptable salts or solvates thereof,of the invention. In other embodiments, the kit comprises one or morepharmaceutical compositions comprising one or more peptides orpharmaceutically acceptable salts or solvates thereof. In certainembodiments, the kit further comprises packaging and/or instructions foruse.

EXAMPLES

The following examples demonstrate certain embodiments of the presentinvention. However, it is to be understood that these examples are forillustration purposes only and do not intend, nor should any beconstrued, to be wholly definitive as to conditions and scope of thisinvention. The examples were carried out using standard techniques,which are well known and routine to those of skill in the art, exceptwhere otherwise described in detail.

Example 1. Nurse Shark Semi-Synthetic Library

For the purposes of the present invention, any method in the art formaking a nurse shark library may be used. In the following examples, anurse shark semi-synthetic library (complexity=1.6×10¹⁰ cfu) wasconstructed. In brief, a vector capable of use in phage display and inmonomeric VNAR expression was constructed. The vector was a modifiedversion of pSEX81 (Progen) in which a 6×His tag (SEQ ID NO: 42), a FLAGtag, and an amber stop codon were inserted between the VNAR sequencesand the full-length PIII protein of the M13 phage. VNAR sequences wereinserted into a restriction site engineered downstream from the PelBsignal sequence of pSEX81 using oligonucleotides by the Quickchangemethod.

Example 2. Selection of BAFF-Binding VNARs

Selection of BAFF-interacting VNARs displayed as a fusion of the PIIIprotein on M13 bacteriophage was performed essentially as described inGriffiths et al. 1994, EMBO J., 13:3245-3260. Briefly, human BAFF(Prospec) was immobilized on Nunc Maxisorp 96-well plates and exposed toan excess (about 100 times the library size) of phages rescued from theOsX-3 library. After incubating for 1.5 hours at room temperature,unbound particles were removed by washing, first in PBS-0.1% Tween, thenin PBS. The bound phages were subsequently eluted with triethylamine(100 mM) and quickly neutralized in Tris (pH=7.5). Eluted particles werethen used to infect E. coli ER2738. A portion of the culture was used toestimate the titer of eluted phages (by counting the number ofantibiotic-resistant colonies), the rest of the culture was infectedwith M13KO7 helper phage to produce phages for the next round ofselection. Four rounds of selection were performed using increasinglystringent conditions consisting in progressively reducing the coatedBAFF concentration at every round (50, 5, 2.5, and 1 μg/mLrespectively), and increasing the washing steps from 10 to 20.

Each of the four input phage populations was tested for specificity toBAFF by polyclonal phage ELISA. Briefly, 10¹² phages were incubated inNunc Maxisorp 96-well plates coated at 1 μg/mL with either BAFF-Fc (SinoBiological) or HSA (Sigma). After incubating for 1 hour at roomtemperature, unbound particles were removed by washing the wells threetimes in PBS-0.1% Tween-20. Bound bacteriophages were then detectedusing a specific anti-M13 antibody (GE) (FIGS. 5-7).

Screening for BAFF-R Binding Clones

After selection rounds three and four, 93 individual clones were pickedand grown in 96 deep-well plates in auto-induction medium (Novagen).Periplasmic protein was extracted by osmotic shock as described inMüller M R et al. (Generation and isolation of target-specificsingle-domain antibodies from shark immune repertoires. Methods MolBiol. 2012; 907:177-94) and directly used in a binding ELISA. Briefly,Nunc Maxisorp 96-well plates were coated at 1 μg/mL with either BAFF-Fcor HSA and periplasmic fraction, pre-blocked in PBS-0.1% Tween+2.5%milk, was exposed to the coated surface. After washing in PBS-0.1%Tween, bound VNARs were detected using a peroxidase-conjugated anti-FLAGantibody (Sigma). Absorbance at 450 nm was recorded using an Envisionmultiwell reader (Perkin Elmer) and BAFF-specific clones were selectedusing the criteria: BAFF signal/HSA signal≥4. The DNA sequence ofpositive clones was determined by the Sanger chain termination method(GATC biotech), using the specific oligonucleotide5′-tcattaggcaccccaggctttacac-3′. Protein alignments were performed usingthe BioEdit software.

Screening for BAFF-R Blocking Clones

The ability of selected BAFF-specific VNARs to prevent the binding ofBAFF to its receptor (BAFF-R) was tested in a blocking ELISA format.Briefly, selected clones were grown in 96 deep-well plates and theperiplasmic fraction was extracted as described above. Nunc Maxisorp96-well plates were coated at 1 μg/mL with the BAFF-R extracellulardomain (Peprotech) and blocked in PBS-0.1% Tween+2.5% milk. Theperiplasmic fraction was also pre-blocked in PBS-0.1% Tween-20+2.5% milkin the presence of 1.14 nM BAFF-Fc (Sino Biological) before beingexposed to the BAFF-R coated surface. After washing in PBS-0.1%Tween-20, BAFF bound to its receptor was detected via its Fc moietyusing a peroxidase-conjugated anti-human Fc (Sigma). Absorbance at 450nm was recorded using an Envision multiwell reader (Perkin Elmer) (FIG.8) and clones reducing the amount of BAFF bound to its receptor by morethan 50% were selected as specific blockers.

Example 3. Expression and Purification of Monomeric BAFF Specific VNARs

Selected BAFF-binding clones were expressed at larger scale in order topurify monomeric VNARs for biochemical analysis. 500 mL-cultures weregrown in auto-induction medium (Novagen) and periplasmic fraction wasextracted by osmotic shock by resuspending the bacteria in TES buffer(50 mM Tris, 1 mM EDTA, 20% Sucrose w/v) mixed with an equal volume ofTES diluted 1:5 in water. After 30 minutes on ice, the lysate wasclarified by centrifugation and the salt concentrations were adjusted to500 mM NaCl and 10 mM imidazole in 1×PBS. The periplasmic fraction wasthen purified on Nickel-Sepharose resin (Qiagen), washed in 1×PBS, 10 mMimidazole, 500 mM NaCl, and then eluted in 1×PBS, 500 mM imidazole, 500mM NaCl. The purified protein was then buffer-exchanged against PBS andconcentrated by centrifugation with Vivaspin 20 filters (Sartorius, MWCO5000). Endotoxin was subsequently removed from the protein sample usingVivaPure Q mini column (Sartorius) and the protein was sterile filtered(0.22 μm). After estimating the protein concentration using Bradfordreagent (Pierce), the purified protein was frozen in aliquots.

Determining Biochemical EC50 Values

The biochemical EC50 (equilibrium constant, the concentration at whichthe ratio of bound to unbound is 50:50) of selected clones wasdetermined by serially diluting purified monomeric VNARs in blockingbuffer (PBS-0.1% Tween+2.5% milk) and exposing it to preblocked NuncMaxisorp 96-well plates coated at 1 μg/mL with BAFF-Fc (SinoBiological). After washing in PBS-0.1% Tween-20, bound VNARs weredetected using a peroxidase-conjugated anti-FLAG antibody (Sigma).Absorbance at 450 nm was recorded using an Envision multiwell reader(Perkin Elmer) (FIG. 9) and EC50s were calculated by fitting curves(non-linear regression) using GraphPad Prism®.

Determining Biochemical IC50 Values

The biochemical IC50 (inhibition constant, the concentration whichinhibits binding of one agent to another agent by 50%) of selectedclones was determined by serially diluting purified monomeric VNARs inblocking buffer (PBS-0.1% Tween-20+2.5% milk) supplemented with 1.14 nMBAFF-Fc. The pre-blocked proteins were then exposed to Nunc Maxisorp96-well plates were coated at 1 μg/mL with the BAFF-R extracellulardomain, preblocked in (PBS-0.1% Tween+2.5% milk). After washing inPBS-0.1% Tween, BAFF bound to its receptor was detected via its Fcmoiety using a peroxidase-conjugated anti-human Fc (Sigma #A0170).Absorbance at 450 nm was recorded using an Envision multiwell reader(Perkin Elmer) (FIG. 10) and IC50 values were calculated by fitting thecurves (non-linear regression) using GraphPad Prism®.

Example 4. Measuring BAFF VNAR Binding Affinities

Surface Plasmon Resonance provides a definitive measure of the affinityof an interaction and may be used to measure affinity of binding by aBAFF binding moiety or BAFF antagonist compound of the invention to aselected target compound, such as human BAFF, mouse or mammaliannon-human BAFF, or a putative cross reactive compound such as APRIL.Specific VNARs of the invention were immobilized on flow cells at adensity of approximately 500RUs. Recombinant BAFF was then applied inthe fluid phase at a flow rate of 20l/min with association for 2minutes, followed by dissociation for 30 minutes at a range of at least6 concentrations from 1 μM to 1 pM. The sensorgrams were then modeled todetermine the kinetic properties of the interaction including rate ofassociation, dissociation and the affinity of the interaction.

Example 5. Murine Splenocyte In Vitro Proliferation Assay

Mouse splenocytes were obtained by dissociating spleens of C57BL/6 miceon a 70 μm cell strainer and lysing red blood cell in RBC buffer(Sigma). B cells were then purified by depleting CD43-positive cellsusing magnetic microbeads (Miltenyi Biotec) according to manufacturer'sinstructions. The obtained B cells were subsequently stimulated withgoat anti-mouse IgM antibody (Jackson Laboratories) at 10 μg/mL finalassay concentration. Recombinant VNARs were serially-diluted andpre-complexed with recombinant BAFF-Fc at 5 ng/mL final assayconcentration in RPMI 1640 supplemented with 10% FBS, for 30 minutes at37° C. Stimulated B cells were added to the pre-complexed proteins andfurther incubated for 72 hours at 37° C., 5% CO₂. Cell proliferation wasthen estimated by incubating cells with WST-1 reagent (Roche) andreading absorbance at 450 nM, subtracting a reference wavelength at 595nM (FIG. 11). IC50 values were calculated by fitting the curves(non-linear regression) using GraphPad Prism®.

Example 6. VNARs that Block the Interaction Between BAFF and TACI orBCMA

The ability of BAFF/BR3 (BAFFr) blocking clones to also block theinteraction between BAFF and its other two receptors (TACI and BCMA)were tested in a blocking ELISA format. Briefly, selected clones weregrown in a 96 deep-well format and periplasmic fractions were extractedas previously described. Nunc Maxisorp 96 well plates were coated at 1μg/mL with either TACI or BCMA (Peprotech) and blocked in PBS-0.1%Tween+2.5% Milk. The periplasmic fraction was also pre-blocked inPBS-0.1% Tween+2.5% Milk in the presence of 0.5 nM BAFF-Fc (SinoBiological) before being exposed to the receptor-coated surface. Afterwashing in PBS-0.1% Tween, BAFF bound to its receptor was detected viaits Fc moiety using a peroxidase-conjugated anti-human Fc (Sigma).Absorbance at 450 nm was recorded using an Envision multiwell reader(Perkin Elmer). The results are shown in FIG. 12. Results showed that ascompared to a negative control VNAR anti-Lysosyme (a-Lys), the thirteentested clones appeared to block the interaction between BAFF and bothTACI and BCMA.

Example 7. VNAR Binding Specificity for BAFF Relative to APRIL

Clones blocking the interaction between BAFF and its three receptorswere tested for their ability to cross-react with BAFF's closest relatedprotein, APRIL. Clones were grown in a 96 well format, periplasmicfraction was extracted by osmotic shock as previously described anddirectly used in a binding ELISA. Nunc Maxisorp 96 well plates werecoated at 1 μg/mL with either BAFF-Fc (Sino Biological), HSA (Sigma), orAPRIL (Sigma) and periplasmic fraction, pre-blocked in PBS-0.1%Tween+2.5% Milk, was exposed to the coated surface. After washing inPBS-0.1% Tween, bound VNARs were detected using a peroxidase-conjugatedanti-FLAG antibody (Sigma). Absorbance at 450 nm was recorded using anEnvision multiwell reader (Perkin Elmer). As shown in FIG. 13, with theexception of B07 which showed a slight binding to APRIL, all the blockerclones appeared specific to BAFF.

Example 8. Epitope Binning of BAFF-Binding VNARs

In order to group the blocking clones into different categories based onthe epitope that each one recognizes on the BAFF proteins, Nunc Maxisorp96 well plates were coated at 1 μg/mL with recombinant BAFF (Sinobiologicals). Clones were grown in a 96 well format and periplasmicfraction was extracted by osmotic shock as previously described. Theperiplasmic fraction was then pre-blocked in PBS-0.1% Tween+2.5% Milk inthe presence of a competitor VNAR-Fc molecule at 2 μM finalconcentration. Two anti-BAFF VNAR-Fcs were used in this assay (A07, B07)as well as a negative control lysozyme-binding VNAR (5A7). Thepre-blocked fraction was then exposed to the coated surface, and afterwashing in PBS-0.1% Tween, bound VNARs were detected using aperoxidase-conjugated anti-FLAG antibody (Sigma). Absorbance at 450 nmwas recorded using an Envision multiwell reader (Perkin Elmer). Theresults are shown in FIG. 14. Results showed that the binding of eachclone to BAFF was competed by both A07 and B07, revealing that all theclones of the selected panel of blockers target the same (or at least anoverlapping) epitope on BAFF.

Example 9. IC50s of Selected VNARs for TACI and BCMA

The biochemical IC50 for each of the five main lead molecules wasdetermined on both TACI and BCMA by serially diluting purified monomericVNARs in blocking buffer (PBS-0.1% Tween+2.5% Milk) supplemented with0.5 nM BAFF-Fc (Sino Biological). The pre-blocked proteins were thenexposed to Nunc Maxisorp 96 well plates which were coated at 1 μg/mLwith the recombinant human TACI or BCMA (Peprotech), preblocked in(PBS-0.1% Tween+2.5% Milk). After washing in PBS-0.1% Tween, BAFF boundto its receptor was detected via its Fc moiety using aperoxidase-conjugated anti-human Fc (Sigma). Absorbance at 450 nm wasrecorded using an Envision multiwell reader (Perkin Elmer) and IC50values were calculated by fitting the curves (non-linear regression)using GraphPad Prism®. The results are shown in FIG. 15 and the fittedIC50 values are tabulated below. Results showed that for the selectedleads, IC50s were generally lower on BCMA than on TACI (compare FIG.15A, TACI coating, to FIG. 15B, BCMA coating).

Fitted IC50 values A07 B07 A05 B10 A09 BR3  6.8 nM  6.8 nM 13.8 nM  2.8nM  9.7 nM TAC I 82.7 nM   24 nM 57.8 nM 14.1 nM 27.2 nM BCMA 24.5 nM18.9 nM 17.4 nM 10.5 nM 23.6 nM

Example 10. Anti-BAFF VNAR Inhibition of Splenocyte Proliferation InVitro

In order to address the species cross-reactivity, the two main leadmolecules (A07 and B07) were expressed and purified as a human Fc fusionmolecule, together with a negative control anti-lysozyme VNAR (5A7), andtested for their ability to block the proliferation of primary mousesplenic B cells stimulated with either mouse or human recombinant BAFF.Mouse splenocytes were obtained by dissociating spleens of C57BL/6 miceon a 70 μm cell strainer and lysing red blood cell in RBC buffer(Sigma). B cells were then purified by depleting CD43-positive cellsusing magnetic microbeads (MACS) according to manufacturer'sinstructions. The obtained B cells were subsequently stimulated withgoat anti-mouse IgM antibody (Jackson) at 10 μg/mL final assayconcentration. Recombinant VNAR-Fcs were serially-diluted andpre-complexed with recombinant human BAFF-Fc (Sino Biological) at 5ng/mL final assay concentration, or recombinant mouse BAFF (R&D Systems)at 0.5 ng/mL final assay concentration, in RPMI 1640 supplemented with10% FBS, for 30 minutes at 37° C. Stimulated B cells were added to thepre-complexed proteins and further incubated for 72 hours at 37° C., 5%CO₂. Cell proliferation was then estimated by incubating cells withWST-1 reagent (Roche) and reading absorbance at 450 nM, subtracting areference wavelength at 595 nM. IC50 values were calculated by fittingthe curves (non-linear regression) using GraphPad Prism®. The resultsare shown in FIG. 16 and the fitted IC50 values are tabulated below.

Fitted IC50 values A07 B07 5A7 BAFFr IC50 (human) 2.48 nM 3.79 nM ~0.066.2 nM IC50 (mouse) 1.95 nM  604 nM ~0.0 7.06 nM

The results showed that both anti-BAFF VNAR molecules werecross-reacting with human and mouse BAFF. Because A07 has asignificantly lower IC50 on mouse BAFF than does B07, it was chosen forsubsequent in vivo mouse studies.

Example 11. Effect of Anti-BAFF VNAR-Fc Splenic B Cell Subsets In Mice

To test the effect of anti-BAFF VNAR-Fc molecules on the development ofB cells in vivo, 12-week-old female C57BL/6 mice were injected IP with100 μg of A07-Fc at days 0 and 5. Mice were sacrificed on day 8, spleenswere extracted and splenocytes were prepared as single cell suspensionsby dispersing the spleens through 100 μM pore size sterile cellstrainers in the presence of DNase at 1 μg/mL. Red blood cells werelysed with Red Blood Cell Lysing Buffer (Sigma). The lysis was carriedout by adding 1 mL of the lysis buffer to cell pellets formed bycentrifugation followed by gentle mixing for 1 minute. The lysis bufferwas then neutralized by adding 19 mL RPMI1640 medium containing 2% heatinactivated fetal calf serum (FCS). The remaining cells were centrifugedagain at 500 g for 7 minutes and supernatants decanted. The pelletedsplenocytes were resuspended in 5 mL of 2% FCS/RPMI1640 medium andpassaged through new sterile cell strainers to remove any aggregates andtotal numbers counted using 0.4% trypan blue. Splenocytes were analysedfor total number of B-lymphocytes and subset frequency by flow cytometryusing the gating scheme described by Scholz and colleagues (Proc NatlAcad Sci USA. 2008 Oct. 7; 105(40): 15517-22). Immature B cells releasedfrom the bone marrow go through transitional stages (classified as T1,T2 & T3 based on the expression of cells surface markers) in the spleenbefore they develop into mature naïve B cells (Mackay and Schneider NatRev Immunol. 2009 July; 9(7):491-502). Results showed that the number oftransitional B cells was significantly reduced in mice treated withA07-Fc as compared to untreated mice (FIG. 17). This effect wasespecially pronounced in late transitional B cells (T3).

Example 12. Effect of Anti-BAFF Formatted on a Mouse Fc Having EffectorFunction

Based on the results of Example 11, the A07 anti-BAFF molecule wasreformatted as a mouse Fc fusion protein with effector function intact.The protein was expressed and purified as described above and EC50binding to human BAFF was assessed by ELISA as previously described. Theresults are shown in FIG. 18. Results showed that A07-mFc displayed anEC50 of about 0.632 nM, while 5A7 did not bind to BAFF. Mice are theninjected i.p. with 100 μg of A07-Fc, 5A7-Fc, or blank control (PBS) onday 0 and again on day 4, 8, and 12 before being be culled on day 15. Bcell subset populations in the spleen are analysed by flow cytometry asdescribed above and improvements in B cell depletion are anticipated.

While some embodiments of the invention have been described by way ofillustration, it will be apparent that the invention can be put intopractice with many modifications, variations and adaptations, and withthe use of numerous equivalents or alternative solutions that are withinthe scope of persons skilled in the art, without departing from thespirit of the invention or exceeding the scope of the claims.

All publications, patents, and patent applications are hereinincorporated by reference in their entirety to the same extent as ifeach individual publication, patent or patent application wasspecifically and individually indicated to be incorporated by referencein its entirety.

What is claimed is:
 1. A method of targeting delivery of a payload to aB-lymphocyte stimulator receptor (BAFF-R)-expressing cell whichcomprises contacting said cell with a BAFF specific bindingmoiety-payload conjugate, wherein said BAFF-specific binding moiety isin a Type 2 shark VNAR-derived scaffold comprising a CDR1 region and aCDR3 region separated by a framework region, wherein the CDR1 regioncomprises or consists essentially of a peptide having an amino acidsequence of formula D-X₂-X₃-X₄-A-L-X₇ wherein X₂ is N or S; X₃ is N, Ior S; X₄ is C or Y; and X₇ is S, P or G (SEQ ID NO: 1); wherein the CDR3region comprises a peptide having an amino acid sequence of formula (a)D-X_(a)-L-Z₍₁₋₆₎-C(SEQ ID NO: 2) or formula (b) C-Z₍₁₋₆₎-D-X_(a)-L (SEQID NO: 3); wherein X_(a) is selected from W, P, R, V or L; whereinZ₍₁₋₆₎ is a stretch of any one to six amino acid residues, with one Zselected from G, L, F or M; and wherein said framework region comprisesFW2-3 of one of SEQ ID NOS: 4-21.
 2. The method of claim 1, wherein theCDR1 region comprises a peptide selected from DNNCALS (SEQ ID NO: 22),DSNCALS (SEQ ID NO: 23), DSNCALP (SEQ ID NO: 24), or DSICALS (SEQ ID NO:25).
 3. The method of claim 1, wherein the CDR3 region comprises apeptide selected from DWLLC (SEQ ID NO: 27), DPLLC (SEQ ID NO: 28),DRLLC (SEQ ID NO: 29), DLLLC (SEQ ID NO: 30), DLLFC (SEQ ID NO: 31),DPLFC (SEQ ID NO: 32), DPLMC (SEQ ID NO: 33), DPLTKEC (SEQ ID NO: 34),DPLLFPRDRC (SEQ ID NO: 36) or HGGRSTGLCGDVLLAGDV (SEQ ID NO: 37).
 4. Themethod of claim 1, wherein the payload is selected from a growth factor,cytokine, lymphokine, cell surface antigen or an antibody or antibodyfragment which binds to any of the foregoing, a chimeric antigenreceptor, a small molecule which is a cytotoxin, a biochemical pathwayagonist or antagonist or a diagnostic agent, or a nucleic acid moleculewith regulatory properties or which encodes a regulatory molecule in acell, or any combination of the foregoing.
 5. The method of claim 4,wherein said diagnostic agent is a fluorescent molecule or othermolecular marker.
 6. The method of claim 1, wherein the BAFF specificbinding moiety-payload conjugate is an immunoconjugate.
 7. The method ofclaim 6, wherein said immunoconjugate is multivalent.
 8. The method ofclaim 6, wherein said immunoconjugate is multispecific.
 9. The method ofclaim 1, wherein said BAFF-R-expressing cell is a B-lymphocyte.
 10. Themethod of claim 9, wherein said B-lymphocyte is malignant.
 11. Themethod of claim 1, wherein said BAFF-R-expressing cell is aT-lymphocyte.
 12. The method of claim 1, wherein delivering said payloadis for treating a disease or condition selected from Hodgkin's lymphoma(HL), Non-Hodgkin's Lymphomas (NHL), Diffuse Large B Cell Lymphoma(DLBCL), Small lymphocytic lymphoma (SLL), Lymphoplasmacytoid lymphoma,Lymphoplasmacytic lymphoma (LPL), Mantle cell lymphoma (MCL), Follicularlymphoma (FL), Marginal zone lymphoma (MZL), Extranodalmucosa-associated lymphoid tissue lymphoma (MALT lymphoma), Nodal(Monocytoid B-cell lymphoma), Splenic Diffuse large cell lymphoma,Burkitt's lymphoma, Lymphoblastic lymphoma, precursor B-lymphoblasticleukemia, B-cell chronic lymphocytic leukemia, chronic lymphocyticleukemia (CLL), acute lymphocytic leukemia (ALL), Plasma cell leukemia,Solitary Plasmacytoma (Bone, Extramedullar), Multiple Myeloma (MM),Smoldering Multiple Myeloma (SMM), non-secretory multiple myeloma, POEMSsyndrome/osteosclerotic myeloma, Monoclonal gammopathy of undeterminedsignificance (MGUS), Waldenstrom's Macroglobulinemia, PrimaryAmyloidosis (AL), Heavy chain disease, Type I and II cryoglobulinemia,Light chain deposition disease, Goodpasture's syndrome, Pemphigus andPemphigoid disorders, Epidermolysis bullosa acquisita, Systemic LupusErythematosus (SLE), Rheumatoid Arthritis (RA), Multiple Sclerosis (MS),Idiopathic Thrombocytopenic Purpura (ITP), Sjogren's syndrome (SS), Type1 or type 2 Diabetes, vasculitis, and graft-vs-host or transplantrejection.
 13. A method of targeting delivery of a payload to aB-lymphocyte stimulator receptor (BAFF-R)-expressing cell whichcomprises contacting said cell with a BAFF specific bindingmoiety-payload conjugate, wherein said BAFF-specific binding moietycomprises a Type 2 shark VNAR of the formulaFW1-CDR1-FW2-FW3-CDR3-FW4, said VNAR comprising any one CDR1 peptide inSEQ ID NOS: 4-21, any one CDR3 peptide in SEQ ID NOS: 4-21, any one FW1peptide in SEQ ID NOS: 4-21, any one FW2-FW3 peptide in SEQ ID NOS: 4-21and any one FW4 peptide in SEQ ID NOS: 4-21.
 14. The method of claim 13,wherein the CDR3 region comprises a peptide having an amino acidsequence selected from RRARVIGGEYCRVQWQDV (SEQ ID NO: 38),LSNVHICCRFGSCADV (SEQ ID NO: 39), APTIISGCSIKRRDV (SEQ ID NO: 40), andTRYVVFSGSTCRMRRADV (SEQ ID NO: 41).
 15. The method of claim 13, whereinsaid VNAR has an amino acid sequence selected from the group consistingof any one of SEQ ID NOS: 4-21.
 16. The method of claim 15, wherein saidVNAR is a variant of a BAFF specific binding moiety which differs by 1to 10 amino acid residues from the recited amino acid sequence andretains BAFF binding activity of at least half of the activity of anon-variant binding moiety.
 17. The method of claim 13, wherein thepayload is selected from a growth factor, cytokine, lymphokine, cellsurface antigen or an antibody or antibody fragment which binds to anyof the foregoing, a chimeric antigen receptor, a small molecule which isa cytotoxin, biochemical pathway agonist or antagonist or a diagnosticagent, or a nucleic acid molecule with regulatory properties or whichencodes a regulatory molecule in a cell, or any combination of theforegoing.
 18. The method of claim 17, wherein said diagnostic agent isa fluorescent molecule or other molecular marker.
 19. The method ofclaim 13, wherein the BAIT specific binding moiety-payload conjugate isan immunoconjugate.
 20. The method of claim 19, wherein saidimmunoconjugate is multivalent.
 21. The method of claim 19, wherein saidimmunoconjugate is multispecific.
 22. The method of claim 13, whereinsaid BAFF-R-expressing cell is a B-lymphocyte.
 23. The method of claim22, wherein said B-lymphocyte is malignant.
 24. The method of claim 13,wherein said BAFF-R-expressing cell is a T-lymphocyte.
 25. The method ofclaim 13, wherein delivering said payload is for treating a disease orcondition selected from Hodgkin's lymphoma (HL), Non-Hodgkin's Lymphomas(NHL), Diffuse Large B Cell Lymphoma (DLBCL), Small lymphocytic lymphoma(SLL), Lymphoplasmacytoid, lymphoma, Lymphoplasmacytic lymphoma, (LPL),Mantle cell lymphoma (MCL), Follicular lymphoma (FL), Marginal zonelymphoma (MZL), Extranodal mucosa-associated lymphoid tissue lymphoma(MALT lymphoma), Nodal (Monocytoid B-cell lymphoma), Splenic Diffuselarge cell lymphoma, Burkitt's lymphoma, Lymphoblastic lymphoma,precursor B-lymphoblastic leukemia, B-cell chronic lymphocytic leukemia,chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL),Plasma cell leukemia, Solitary Plasmacytoma (Bone, Extramedullar),Multiple Myeloma (MM), Smoldering Multiple Myeloma (SMM), non-secretorymultiple myeloma, POEMS syndrome/osteosclerotic myeloma, Monoclonalgammopathy of undetermined significance (MGUS), Waldenstrom'sMacroglobulinemia, Primary Amyloidosis (AL), Heavy chain disease, Type Iand II cryoglobulinemia, Light chain deposition disease, Goodpasture'ssyndrome, Pemphigus and Pemphigoid disorders, Epidermolysis bullosaacquisita, Systemic Lupus Erythematosus (SLE), Rheumatoid Arthritis(RA), Multiple Sclerosis (MS), Idiopathic Thrombocytopenic Purpura(ITP), Sjogren's syndrome (SS), Type 1 or type 2 Diabetes, vasculitis,and graft-vs-host or transplant rejection.